CN111132697A - Pharmaceutical combination comprising anti-BST-1 antibodies and cytidine analogs - Google Patents

Abstract

The present disclosure relates to a pharmaceutical combination comprising an antibody against BST1 (ADP-ribosyl cyclase 2) and a cytidine analog or a pharmaceutically acceptable salt thereof; and methods for treating diseases such as cancer mediated by BST1 (ADP-ribosyl cyclase 2) expression/activity and/or associated with aberrant expression/activity of BST 1.

Description

Pharmaceutical combination comprising anti-BST-1 antibodies and cytidine analogs

Introduction to the word

The present disclosure relates generally to the fields of immunology and molecular biology. More specifically, provided herein are pharmaceutical combinations comprising an antibody against BST1 (ADP-ribosyl cyclase 2) and a cytidine analog or a pharmaceutically acceptable salt thereof; and methods for treating diseases such as cancer mediated by BST1 (ADP-ribosyl cyclase 2) expression/activity and/or associated with aberrant expression/activity of BST 1.

Background

Leukemias and lymphomas belong to a large group of tumors that affect the blood, bone marrow, and lymphatic system; these tumors are known as hematopoietic and lymphoid tumors.

Lymphomas are a group of blood cell tumors that develop from lymphocytes. Signs and symptoms include swollen lymph nodes, fever, profuse sweating, involuntary weight loss, itching and continuous feeling of tiredness. Lymphomas exist in several subtypes: two major classes of lymphoma are Hodgkin's Lymphoma (HL) and non-Hodgkin's lymphoma (NHL). The World Health Organization (WHO) includes two other classes of lymphoma types: multiple myeloma and immunoproliferative diseases. About 90% of lymphomas are non-hodgkin lymphomas.

Leukemia is a group of cancers that usually begin in the bone marrow and result in large numbers of abnormal white blood cells. Symptoms may include bleeding and bruising problems, feeling tired, fever and increased risk of infection. These symptoms occur due to the lack of normal blood cells. Diagnosis is usually performed by blood tests or bone marrow biopsies. There are four major leukemia types: acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), and Chronic Myeloid Leukemia (CML), as well as a variety of less common types.

The treatment of leukemia and lymphoma involves one or more of the following: chemotherapy, radiation therapy, targeted therapy and surgery (and bone marrow transplantation in the case of leukemia). The success of leukemia therapy depends on the type of leukemia and the age of the individual. Lymphoma treatment results depend on subtype, some are curable and treatment can prolong survival in most cases.

A variety of chemotherapeutic agents have previously been used to treat leukemia, including prednisone (prednisone), vincristine, anthracyclines, L-asparaginase, cyclophosphamide, methotrexate, 6-mercaptopurine, fludarabine (fludarabine), pentostatin (pentostatin), and cladribine (cladribine). Chemotherapeutic agents used to treat lymphoma include cyclophosphamide, hydroxydaunorubicin (also known as doxorubicin (doxorubicin) or adriamycin (adriamycin)), ancovenin (oncovin), prednisone, prednisolone (prednisolone), bleomycin (bleomycin), dacarbazine (dacarbamesine), etoposide (etoposide), and procarbazine (procarbazine).

Combination chemotherapy involves treating a patient with two or more different drugs simultaneously. Drugs may differ in their mechanism and side effects. This is most advantageous in that it minimizes the possibility of developing resistance to either agent. In addition, drugs can often be used at lower doses, reducing toxicity. Combination therapy for the treatment of hodgkin's disease includes MOPP (nitrogen mustard, vincristine, procarbazine, prednisolone) and ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine). Combination therapies for the treatment of non-hodgkin's lymphoma include CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone). The number of permutations and combinations of possible drug therapies is obviously large, considering the number of drugs known for the treatment of leukemias and lymphomas. Furthermore, the aforementioned combination therapies do not include antibodies.

However, there is still a need for new treatments, in particular effective combination therapies, for leukemia and lymphoma.

Bone marrow stromal antigen 1(BST1), also known as ADP-ribosyl cyclase 2 or CD157, is a lipid-anchored bifunctional extracellular enzyme that catalyzes ribonucleotide cyclization and hydrolysis. It produces nucleotides, second messenger circular ADP-ribose and ADP-ribose, capable of activating calcium release and protein phosphorylation (FEBS Lett.1994,356(2-3): 244-8). It is able to support the growth of pre-B cells in a paracrine fashion, possibly through the production of NAD + metabolites (Proc. Natl. Acad. Sci. USA 1994,91: 5325-.

ADP-ribosyl cyclase 2 and its homologue CD38 appear to act as a receptor, producing second messenger metabolites that induce intracellular Ca2+ release via the ryanodine receptor (biochem. Biophys. Res. Commun.1996,228(3): 838-45). It may also act via CD11b integrin to achieve Ca2+ release via the PI-3 kinase pathway (j.biol.regul.homeost.Agents.2007; 21(1-2): 5-11).

WO2013/003625 discloses anti-BST 1 antibodies and their use for treating various cancers.

5-aza-cytidine and 5-aza-2' -deoxycytidine are cytidine analogs currently used to treat myeloma or myelodysplastic syndrome.

It has now been found that the combination of (i) certain anti-BST 1 antibodies with 5-aza-cytidine and (ii) certain anti-BST 1 antibodies with 5-aza-2' -deoxycytidine exhibits synergistic results in the treatment of leukemia and other cancers associated with BST1 expression.

Disclosure of Invention

The present invention provides a pharmaceutical combination comprising (a) an antibody against BST1 and (B) a cytidine analog, or a pharmaceutically acceptable salt thereof; and methods for treating diseases, such as BST 1-mediated disorders, e.g., human cancers, including Acute Myeloid Leukemia (AML), B-cell chronic lymphocytic leukemia, breast cancer, colorectal cancer, renal cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, hereinafter referred to as "the diseases of the invention".

In one embodiment, the pharmaceutical combination comprises:

(A) an anti-BST 1 antibody or antigen-binding portion thereof that competes for binding to BST1 with an antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 2 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 4;

or an isolated anti-BST 1 antibody, or antigen-binding portion thereof, comprising:

a) a heavy chain variable region comprising:

i) a first CDR comprising a sequence at least 80% identical to SEQ ID NO 10;

ii) a second CDR comprising a sequence at least 80% identical to SEQ ID NO 12 or SEQ ID NO 51;

iii) a third CDR comprising a sequence at least 80% identical to SEQ ID NO 14; and

b) a light chain variable region comprising:

I) a first CDR comprising a sequence at least 80% identical to SEQ ID NO 16;

ii) a second CDR comprising a sequence at least 80% identical to SEQ ID NO 18;

iii) a third CDR comprising a sequence at least 80% identical to SEQ ID NO 20;

still further wherein any one or more of the above SEQ ID NOs each independently comprise one, two, three, four or five amino acid substitutions, additions or deletions;

and

(B) a cytidine analog, or a pharmaceutically acceptable salt thereof,

wherein the pharmaceutical combination is in the form of a combined preparation for simultaneous, separate or sequential use, preferably in the treatment of cancer.

Preferably, the cytidine analog is 5-aza-cytidine or 5-aza-2' -deoxycytidine. 5-aza-cytidine is a chemical analog of cytidine, a nucleoside in DNA and RNA. It has the structure:

5-aza-cytidine is also known under the trade names Vidaza and Azadine.

5-aza-2' -deoxycytidine is also a chemical analogue of cytidine. It has the structure:

5-aza-2' -deoxycytidine is also known as Decitabine (Decitabine) and is known under the trade name Dacogen.

The epitope recognized by the antibody of the present invention is present within the polypeptide sequence of SEQ ID NO. 44.

In yet another embodiment, the isolated anti-BST 1 antibody has a heavy chain variable region sequence as represented by SEQ ID NO. 2 and a light chain variable region sequence as represented by SED ID NO. 4.

In another embodiment, the isolated anti-BST 1 antibody has a heavy chain variable region sequence as represented by SEQ ID NO:46 and a light chain variable region sequence as represented by SED ID NO: 49.

In one embodiment, any of the foregoing antibodies possesses an Fc domain. In some embodiments, the Fc domain is human. In other embodiments, the Fc domain is a variant human Fc domain.

In another embodiment, any of the foregoing antibodies is a monoclonal antibody.

In one embodiment, any of the foregoing antibodies is further provided with a conjugated reagent. In some embodiments, the conjugated agent is a cytotoxic agent. In other embodiments, the coupling agent is a polymer. In another embodiment, the polymer is polyethylene glycol (PEG). In another embodiment, the PEG is a PEG derivative.

In one embodiment, the isolated antibody is an antibody that competes with BST1_ a2 for binding to BST 1.

Any of the anti-BST 1 antibodies can be provided in a pharmaceutical composition.

In another embodiment, the present invention provides a method for the treatment or prevention of a disease associated with BST1 or with target cells expressing BST1, preferably cancer, more preferably human cancer, comprising administering to a subject in need thereof simultaneously, sequentially or separately therapeutically effective amounts of components (a) and (B) of the pharmaceutical combination of the present invention.

In some embodiments, the antibody is a full length antibody of the IgG1, IgG2, IgG3, or IgG4 isotype.

In some embodiments, the antibody is selected from the group consisting of: whole antibodies, antibody fragments, humanized antibodies, single chain antibodies, immunoconjugates, defucosylated antibodies, and bispecific antibodies. The antibody fragment may be selected from the group consisting of: UniBody, domain antibody, and Nanobody. In some embodiments, the immunoconjugate of the invention comprises a therapeutic agent. In another aspect of the invention, the therapeutic agent is a cytotoxin or a radioisotope.

In some embodiments, the antibody is selected from the group consisting of: affibody (Affibody), DARPin, Anticalin (Anticalin), Avimer, Versabody, and Duocalin.

In other embodiments, the pharmaceutical combination of the invention comprises an antibody, or antigen-binding portion thereof, and a pharmaceutically acceptable carrier.

In some embodiments, the invention comprises a kit comprising one or more expression vectors comprising an isolated nucleic acid molecule encoding a heavy chain and/or a light chain of an antibody, or antigen-binding portion thereof, that binds to an epitope on human BST 1; and cytidine analogs, preferably 5-aza-cytidine or 5-aza-2' -deoxycytidine, or pharmaceutically acceptable salts thereof.

In other embodiments, the invention relates to the pharmaceutical combination of the invention for use in the treatment or prevention of a disease associated with target cells expressing BST 1. In some aspects, the disease treated or prevented is cancer, preferably human cancer. In some embodiments, the disease treated or prevented by an antibody of the invention is a disease of the invention.

In other embodiments, the present invention relates to the use of components (a) and (B) of the pharmaceutical combination of the present invention for the preparation of a pharmaceutical combination for simultaneous, separate or sequential use in the treatment or prevention of a disease associated with target cells expressing BST 1. In some aspects, the disease treated or prevented is cancer, preferably human cancer.

In a preferred embodiment, the pharmaceutical combination of the invention is useful for the treatment or prevention of Acute Myeloid Leukemia (AML), B-cell chronic lymphocytic leukemia, breast cancer, colorectal cancer, kidney cancer, head and neck cancer, lung cancer, ovarian cancer and/or pancreatic cancer, preferably Acute Myeloid Leukemia (AML).

In some aspects of the invention, the antibody, or antigen-binding portion thereof, binds to an epitope on a BST1 polypeptide having the amino acid sequence of SEQ ID No. 44 that is recognized by an antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 2 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 4.

Other features and advantages of the present invention will be apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, Genbank entries, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

Drawings

FIG. 1a shows the nucleotide sequence of the heavy chain CDR1 region of A1 (SEQ ID NO:21) in combination with mouse germline V_HAlignment of nucleotides 138392-138424 of the nucleotide sequences 1-80 (SEQ ID NO: 33); nucleotide sequence of heavy chain CDR1 region of A2 (SEQ ID NO:22) and mouse germline V_HAlignment of nucleotides 153362-153394 of the nucleotide sequences 1-39 (SEQ ID NO: 35).

FIG. 1b shows the nucleotide sequence of the heavy chain CDR2 region of A1 (SEQ ID NO:23) vs. mouse germline V_H1-80 nucleotide sequence of nucleotides 138461-138511(SEQ ID NO: 34); nucleotide sequence of heavy chain CDR2 region of A2 (SEQ ID NO:24) and mouse germline V_HAlignment of nucleotides 153431-153481 of the nucleotide sequences 1-39 (SEQ ID NO: 36).

FIG. 2a shows the nucleotide sequence of the light chain CDR1 region of A1 (SEQ ID NO:27) vs mouse germline V_KAlignment of nucleotides 496-531(SEQ ID NO:37) of the nucleotide sequences 4 to 74; nucleotide sequence of light chain CDR1 region of A2 (SEQ ID NO:28) and mouse germline V_KAlignment of nucleotide 523-552(SEQ ID NO:40) of the 4-55 nucleotide sequences.

FIG. 2b shows the nucleotide sequence of the light chain CDR2 region of A1 (SEQ ID NO:29) in combination with mouse germline V_KNucleotide 577-597 of the nucleotide sequence 4-74 (SEQ ID NO: 38)) Comparing; alignment of the nucleotide sequence of the light chain CDR2 region of A2 (SEQ ID NO:30) with nucleotide 598-618(SEQ ID NO:41) of the mouse germline VK 4-55 nucleotide sequence.

FIG. 2c shows an alignment of the nucleotide sequence of the light chain CDR3 region of A1 (SEQ ID NO:31) with nucleotides 691-718(SEQ ID NO:39) of the mouse germline VK 4-74 nucleotide sequence; alignment of the nucleotide sequence of the light chain CDR3 region of A2 (SEQ ID NO:32) with nucleotide 715-739(SEQ ID NO:42) of the mouse germline VK 4-55 nucleotide sequence.

Fig. 3a and 3b show the results of flow cytometric analysis of BST1 on a549 and H226 cells.

Figures 4a and 4b show internalization of anti-BST 1 monoclonal antibodies by a549 and H226 cells using the MabZAP assay.

FIG. 5 shows residues 21-137 of SEQ ID NO: 2(SEQ ID NO:45), CDR regions of SEQ ID NO:2 (highlighted in bold) transfer to human germline BF238102V_HHumanized V of corresponding position_HChain (SEQ ID NO:46) and human germline BF238102V_H(SEQ ID NO: 47). Residues showing significant contact with the CDR regions were substituted for the corresponding human residues. These substitutions (underlined) are made at positions 30, 48, 67, 71 and 100.

FIG. 6 shows the transfer of residues 22-128 of SEQ ID NO: 4(SEQ ID NO:48), the CDR regions of SEQ ID NO:4 (highlighted in bold) to human germline X72441V_LHumanized V of corresponding position_LChain (SEQ ID NO:49) and human germline X72441V_L(SEQ ID NO: 50). Residues showing significant contact with the CDR regions were substituted for the corresponding human residues. One substitution (underlined) was made at position 71.

FIG. 7 shows an alignment of the CDR2 region of the heavy chain of A2 (SEQ ID NO:12) with possible amino acid substitutions (SEQ ID NO:51) without loss of antigen binding affinity.

Figure 8a shows that BST1_ a2 and BST1_ a2_ NF elicits antibody-dependent cellular cytotoxicity (ADCC) responses in the presence of effector cells.

Figure 9 shows the level of ADCC induced by BST1_ a2+ 5-azacytidine in K052 cells.

Figure 10 shows ADCC levels induced by BST1_ a2+ 5-azacytidine in SKNO1 cells.

Figure 11 shows ADCC levels induced by BST1_ a2+ decitabine in SKNO1 cells.

Figure 12 shows ADCC levels induced by BST1_ a2+ decitabine in HL60 cells.

Detailed Description

In order that the invention may be more readily understood, certain terms are first defined. Other definitions are given in the detailed description.

In certain instances, the humanized and murine antibodies described herein may cross-react with BST1 from species other than humans. In certain embodiments, the antibodies are fully specific for one or more human BST1 and do not exhibit species or other types of non-human cross-reactivity.

The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or liver (including antibodies, cytokines, and complement) that results in the selective damage, destruction, or elimination from the human body of invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the context of autoimmunity or pathological inflammation.

"Signal transduction pathway" refers to the biochemical relationship between various signal transduction molecules that function in the transmission of signals from one part of a cell to another. As used herein, the phrase "cell surface receptor" includes, for example, molecules and molecular complexes that are capable of receiving a signal and transmitting such signal across the plasma membrane of a cell. An example of a "cell surface receptor" is BST 1.

The term "antibody" as used herein includes at least an antigen-binding fragment (i.e., "antigen-binding portion") of an immunoglobulin.

The definition of "antibody" includes, but is not limited to, full length antibodies, antibody fragments, single chain antibodies, bispecific antibodies, minibodies (minibodies), domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and the corresponding respective of the foregoingA fragment and/or derivative of (a). In general, a full-length antibody (sometimes referred to herein as a "whole antibody") refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains C _H1、C _H2 and C _H3. Each light chain comprises a light chain variable region (abbreviated herein as V)_LOr V_K) And a light chain constant region. The light chain constant domain comprises a domain C_L。V_HRegion and V_L/V_KRegions can be further divided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions, termed Framework Regions (FRs). Each V_HAnd V_L/V_KComprising three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

In one embodiment, the antibody is an antibody fragment. Specific antibody fragments include, but are not limited to, (i) a fragment consisting of V_L、V_H、C_LAnd C _H1 domain; (ii) from V_HAnd C _H1 domain; (iii) v from a single antibody_LAnd V_H(iii) an Fv fragment consisting of a domain; (iv) a dAb fragment consisting of a single variable domain; (v) an isolated CDR region; (vi) a F (ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single chain Fv molecule (scFv), wherein V_HDomains and V_LThe domains are linked by a peptide linker that allows the two domains to associate to form an antigen binding site; (viii) bispecific single chain Fv dimers; and (ix) "diabodies" or triabodies ", multivalent or multispecific fragments constructed by gene fusion. Antibody fragments may be modified. For example, by including a connection V_HAnd V_LTwo of the structural domainsThe sulfur bond stabilizes the molecule. Examples of antibody formats and constructs are described in Holliger and Hudson (2006) Nature Biotechnology23(9):1126-1136 and Carter (2006) Nature Reviews Immunology6:343-357, and references cited therein, all of which are expressly incorporated by reference.

The present invention provides antibody analogs. The analogs can comprise a variety of structures, including, but not limited to, full-length antibodies, antibody fragments, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), antibody fusions, antibody conjugates, and the respective corresponding fragments of the foregoing.

In one embodiment, the immunoglobulin comprises an antibody fragment. Specific antibody fragments include, but are not limited to, (i) a fragment consisting of V_L、V_H、C_LAnd C _H1 domain; (ii) from V_HAnd C _H1 domain; (iii) v from a single antibody_LAnd V_H(iii) an Fv fragment consisting of a domain; (iv) a dAb fragment consisting of a single variable domain; (v) an isolated CDR region; (vi) a F (ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single chain Fv molecule (scFv), wherein V_HDomains and V_LThe domains are linked by a peptide linker that allows the two domains to associate to form an antigen binding site; (viii) bispecific single chain Fv dimers; and (ix) "diabodies" or "triabodies", multivalent or multispecific fragments constructed by gene fusion. Antibody fragments may be modified. For example, by including a connection V_HAnd V_LDisulfide bonds of the domains stabilize the molecule. Examples of antibody formats and constructs are described in Holliger and Hudson,2006, Nature Biotechnology23(9):1126-1136 and Carter,2006, Nature Reviews Immunology6:343-357 and references cited therein, all of which are expressly incorporated by reference.

Recognized immunoglobulin genes (e.g., in humans) include kappa (kappa), lambda (lambda) and heavy chain loci, which together comprise myriad variable region genes, and constant region genes mu (mu), delta (delta), gamma (gamma), sigma (sigma) and alpha (sigma), which encode IgM, IgD, IgG (IgG1, IgG2, IgG3 and IgG4), IgE and IgA (IgA1 and IgA2) isotypes, respectively. Antibodies herein are meant to include full length antibodies and antibody fragments, and may refer to natural antibodies, engineered antibodies, or recombinantly produced antibodies from any organism for experimental, therapeutic, or other purposes.

In one embodiment, the antibodies disclosed herein can be multispecific antibodies, and notably bispecific antibodies, sometimes referred to as "diabodies". They are antibodies that bind to two (or more) different antigens. Diabodies can be prepared in a variety of ways known in the art, e.g., chemically or from hybridomas. In one embodiment, the antibody is a minibody. Minibodies are minimized antibody-like proteins comprising a peptide with C _H3 domain linked scFv. In some cases, the scFv may be linked to the Fc region and may comprise some or all of the hinge region. For a description of multispecific antibodies, see Holliger and Hudson (2006) Nature Biotechnology23(9):1126 and 1136 and references cited therein, all of which are expressly incorporated by reference.

As used herein, "CDR" means the "complementarity determining region" of an antibody variable domain. Systematic identification of residues included in CDRs has been developed by Kabat (Kabat et al, (1991) protein Sequences of Immunological Interest (Sequences of proteins of Immunological Interest), 5 th edition, United states public Health Service (United states public Health Service), National Institutes of Health (National Institutes of Health), Besserda) and alternatively by Chothia [ Chothia and Lesk (1987) J.mol.biol.196: 901-; chothia et al, (1989) Nature 342: 877-883; Al-Lazikani et Al, (1997) J.mol.biol.273:927-948]. For the purposes of the present invention, a CDR is defined as a slightly smaller set of residues than the CDR defined by Chothia. V_LCDRs are defined herein as including residues at positions 27-32(CDR1), 50-56(CDR2), and 91-97(CDR3), where numbering is according to Chothia. Because of V as defined by Chothia and Kabat_LCDRs are identical, so these V_LThe numbering of the CDR positions is also according to Kabat. V_HCDRs are defined herein as being included at positions 27-33(CDR1), 52-56(CDR2) and 95-10Residue at 2(CDR3), where numbering is according to Chothia. These V_HThe CDR positions correspond to Kabat positions 27-35(CDR1), 52-56(CDR2) and 95-102(CDR 3).

One skilled in the art will appreciate that the CDRs disclosed herein may also include variants, for example, when the CDRs disclosed herein are back mutated (back mutate) into different framework regions. Typically, the nucleic acid identity between individual variant CDRs is at least 80% relative to the sequences described herein, and more typically preferably increased by at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and almost 100% identity. Similarly, "percent (%) nucleic acid sequence identity" with respect to the nucleic acid sequence of a binding protein identified herein is defined as: the percentage of nucleotide residues in the candidate sequence that are identical to the nucleotide residues in the antigen binding protein coding sequence. A particular method uses the BLASTN module of WU-BLAST-2 set to preset parameters and sets the overlap span (overlap span) and the overlap fraction (overlap fraction) to 1 and 0.125, respectively, and no filter is selected.

Typically, the nucleic acid sequence identity between a nucleotide sequence encoding an individual variant CDR and a nucleotide sequence described herein is at least 80%, and more typically preferably increased by at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and almost 100% identity.

Thus, a "variant CDR" is a CDR that has a specified homology, similarity, or identity to a parent CDR of the invention and shares a biological function, including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% specificity and/or activity with the parent CDR.

Although the site or region for introducing an amino acid sequence variation is predetermined, the mutation itself need not be predetermined. For example, to optimize the performance of a mutation at a given site, random mutagenesis can be performed at the target codon or region and the expressed antigen binding protein CDR variants screened for the optimal combination of desired activities. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis. Screening of mutants was performed using the antigen binding protein activity assay as described herein.

Amino acid substitutions are typically single residues; insertions are typically on the order of about one (1) to about twenty (20) amino acid residues, although significantly larger insertions can be tolerated. Deletions range from about one (1) to about twenty (20) amino acid residues, although in some cases the deletion may be larger.

Substitutions, deletions, insertions or any combination thereof may be used to arrive at the final derivative or variant. Typically, these changes are made at a number of amino acids to minimize the alteration of the molecule, particularly the immunogenicity and specificity of the antigen binding protein. However, in some cases larger changes can be tolerated.

As used herein, "Fab" or "Fab region" is meant to encompass V_H、C _H1、V_LAnd C_LA polypeptide of an immunoglobulin domain. Fab may refer to this region isolated, or in the context of a full-length antibody, antibody fragment, or Fab fusion protein, or any other antibody embodiment as outlined herein.

As used herein, "Fv" or "Fv fragment" or "Fv region" is meant to encompass a single antibody V_LAnd V_HA polypeptide of domain.

As used herein, "framework" means a region that does not comprise antibody variable domains defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions separated by CDRs (FR1, FR2, FR3 and FR 4).

As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") is one or more antibody fragments that retain the ability to specifically bind to an antigen (e.g., BST 1). It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) a Fab fragment, consisting of V_L/V_K、V_H、C_LAnd C _H1 domain; (ii) f (ab')₂A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; (iii) fab' fragments, essentially Fab with a partial hinge region (see "basic IMMUNOLOGY" (FUNDAMENTAL IMMUNOLOGY) (Paul eds., 3 rd supplementary revision, 1993); (iv) V_HAnd C _H1 domain; (v) v with one arm consisting of antibody_LAnd V_H(iii) an Fv fragment consisting of a domain; (vi) from V_HdAb fragments consisting of domains [ Ward et al (1989) Nature 341:544-546](ii) a (vii) An isolated Complementarity Determining Region (CDR); and (viii) nanobodies, heavy chain variable regions comprising a single variable domain and two constant domains. In addition, although two domains of the Fv fragment, V_L/V_KAnd V_HEncoded by separate genes, but can be joined using recombinant methods by synthetic linkers that enable their production as single protein chains in which V is present_L/V_KAnd V_HThe regions pair to form monovalent molecules (referred to as single chain fv (scFv); see, e.g., Bird et al (1988) Science 242: 423-. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques well known to those skilled in the art, and the fragments are screened for use in the same manner as whole antibodies.

As used herein, "isolated antibody" means an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds BST1 is substantially free of antibodies that specifically bind antigens other than BST 1). However, isolated antibodies that specifically bind BST1 can be cross-reactive to other antigens (such as BST1 molecules from other species). Additionally and/or alternatively, the isolated antibody can be substantially free of other cellular material and/or chemicals in forms not normally found in nature.

In some embodiments, the antibodies of the invention are recombinant proteins, isolated proteins, or substantially pure proteins. An "isolated" protein is not accompanied by at least some of the substances with which it is normally associated in its native state, e.g., constitutes at least about 5% or at least about 50% by weight of the total protein in a given sample. It is understood that the isolated protein may constitute from 5% to 99.9% by weight of the total protein content, as the case may be. For example, a protein can be produced at significantly higher concentrations through the use of inducible promoters or high expression promoters, thereby producing the protein at increased concentration levels. In the case of recombinant proteins, this definition includes the production of antibodies in a wide variety of organisms and/or host cells known in the art in which antibodies are not naturally produced.

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. As used herein, "polyclonal antibodies" refers to antibodies produced by several clones of B-lymphocytes, as is the case in intact animals.

As used herein, "isotype" refers to the class of antibodies (e.g., IgM or IgG1) encoded by the heavy chain constant region genes.

The phrases "an antibody that recognizes an antigen" and "an antibody having specificity for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen".

The term "antibody derivative" refers to any modified form of an antibody, e.g., a conjugate (usually a chemical linkage) of an antibody to another agent or antibody. For example, the antibodies of the invention can be conjugated to agents, including but not limited to polymers (e.g., PEG), toxins, labels, and the like, as described in more detail below. The antibodies of the invention may be non-human, chimeric, humanized or fully human. For a description of the concept of chimeric and humanized antibodies, see Clark et al (2000) and references cited therein (Clark,2000, immunological Today21: 397-. Chimeric antibodies comprise a non-human variable region operatively linked to a human constant region, e.g., a mouse or rat V_HAnd V_LDomains (see, e.g., U.S. Pat. No.)No. 4,816,567). In a preferred embodiment, the antibody of the invention is humanized. As used herein, a "humanized" antibody is intended to mean an antibody that comprises a framework-like region (FR) and one or more Complementarity Determining Regions (CDRs) from a non-human (typically mouse or rat) antibody. The non-human antibody that provides the CDRs is referred to as the "donor" and the human immunoglobulin that provides the framework is referred to as the "acceptor". Humanization relies in principle on grafting donor CDRs to recipient (human) V_LAnd V_HOn the frame (U.S. patent No. 5,225,539). This strategy is called "CDR grafting". It is often desirable to "back-mutate" selected acceptor framework residues to the corresponding donor residues to regain the affinity lost in the originally transplanted construct (U.S. Pat. No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat. No. 5,821,337; U.S. Pat. No. 6,054,297. Preferably, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region, typically the corresponding portion of a human immunoglobulin, and thus will typically comprise a human Fc region. Methods for humanizing non-human antibodies are well known in the art and can be performed essentially following the method of Winter and co-workers [ Jones et al (1986) Nature 321: 522-525; riechmann et al (1988) Nature332: 323-329; verhoeyen et al (1988) Science,239:1534-]. Other examples of humanized murine monoclonal antibodies are also known in the art, e.g., binding to human Protein C (O' Connor et al, 1998, Protein Eng11:321-8), interleukin 2 receptor [ Queen et al (1989) Proc Natl Acad Sci, USA 86:10029-33]And human epidermal growth factor receptor 2[ Carter et al (1992) Proc Natl Acad Sci USA 89:4285-9]The antibody of (1). In another embodiment, the antibody of the invention may be a fully human antibody, i.e., the antibody sequence is fully or substantially human. A number of methods are known in the art for the production of fully human antibodies, including the use of transgenic mice [ Bruggemann et al (1997) Curr Opin Biotechnol 8: 455-458-]Or human antibody libraries together with a selection method [ Griffiths et al (1998) Curr Opin Biotechnol 9: 102-108-]。

The term "humanized antibody" is intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences. Additional framework region modifications, such as Fc domain amino acid modifications, can be made within the human framework sequences, as described herein.

The term "chimeric antibody" means an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

The term "specific binding" (or "immunospecific binding") is not intended to mean that an antibody binds only to its target of interest, although this is the case in many embodiments; that is, the antibody "specifically binds" to its target and non-detectably binds or does not substantially bind to other components in the sample, cell, or patient. However, in some embodiments, an antibody "specifically binds" if the affinity of the antibody for its target is about 5-fold when compared to the affinity of the antibody for non-target molecules. Suitably, there is no significant cross-reaction or cross-binding with unwanted substances, especially proteins or tissues naturally present in healthy humans or animals. For example, the affinity of an antibody for a target molecule will be at least about 5-fold, such as 10-fold, such as 25-fold, especially 50-fold and especially 100-fold or more greater than its affinity for a non-target molecule. In some embodiments, specific binding between an antibody or other binding agent and an antigen means at least 10⁶M^-1Binding affinity of (4). For example, the antibody can be at least about 10⁷M^-1Such as about 10⁸M^-1To about 10⁹M^-1About 10⁹M^-1To about 10¹⁰M^-1Or about 10¹⁰M^-1To about 10¹¹M^-1Affinity binding between. For example, the antibody may have an EC of 50nM or less, 10nM or less, 1nM or less, 100pM or less, or more preferably 10pM or less₅₀And (4) combining.

As used herein, the term "substantially not bind" to a protein or cell means not binding or binding with high affinity to a protein or cell, i.e. at 1x10^-6M or greater, preferably 1x10^-5M or greater, preferably 1x10^-4M or greater, more preferably 1x10^-3M or greater, even more preferably 1x10^-2K of M or greater_DBinding to proteins or cells.

As used herein, the term "EC₅₀By "is meant the potency of a compound as determined by quantifying the concentration that results in 50% of the maximal response/effect. EC determination by Scatchard or FACS₅₀。

As used herein, the term "K_assoc"or" K_a"means the binding rate of a particular antibody-antigen interaction, and as used herein, the term" K_dis"or" K_dBy "is meant the off-rate of a particular antibody-antigen interaction. As used herein, the term "K_D"means the affinity constant, which is defined by the ratio of Kd to Ka (i.e., K)_d/K_a) Obtained and expressed as molar concentration (M). The K of an antibody can be determined using well established methods in the art_DThe value is obtained. Method for determining antibody K_DPreferably using surface plasmon resonance, preferably using biosensor systems such as

Provided is a system.

As used herein, the term "high affinity" for an IgG antibody means that the antibody has 1x10 for the target antigen^-7M or less, more preferably 5x10^-8M or less, even more preferably 1x10^-8M or less, even more preferably 5x10^-9M or less, and even more preferably 1x10^-9M or less KD. However, "high affinity" binding may vary with other antibody isotypes. For example, "high affinity" binding for an IgM isotype means that the antibody has 10^-6M or less, more preferably 10^-7M or less, even more preferably 10^-8M or less KD.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed of contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from consecutive amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3,4, 5,6, 7, 8, 9,10, 11, 12, 13, 14 or 15 amino acids in a unique spatial configuration. Methods for determining the spatial configuration of an Epitope include techniques known in the art and those described herein, such as x-ray crystallography and 2-dimensional nuclear magnetic resonance [ see, e.g., Epitope mapping protocol in Methods of Molecular Biology (Epitope mapping protocols in Methods in Molecular Biology), Vol.66, eds. G.E.Morris (1996) ].

Thus, the invention also includes pharmaceutical combinations comprising antibodies that bind to (i.e., recognize) the same epitope as BST1_ a 2. Antibodies that bind to the same epitope can be identified by their ability to cross-compete with the reference antibody for the target antigen in a statistically significant manner (i.e., competitively inhibit the binding of the reference antibody to the target antigen). For example, competitive inhibition can occur if an antibody binds to the same or structurally similar epitope (e.g., an overlapping epitope) or to a spatially proximal epitope that causes steric hindrance between the antibodies upon binding.

Competitive inhibition can be determined using routine assays in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen. Numerous types of competitive binding assays are known, for example: solid phase direct or indirect Radioimmunoassay (RIA), solid phase direct or indirect Enzyme Immunoassay (EIA), mixed competition assay [ see Stahl et al (1983) Methods in Enzymology 9:242 ]; solid phase direct biotin-avidin EIA [ see Kirkland et al (1986) J.Immunol.137:3614 ]; solid phase direct labeling assay, solid phase direct labeling mix assay [ see Harlow and Lane (1988) handbook of antibody laboratories (Antibodies: A Laboratory Manual), Cold Spring Harbor Press (Cold Spring Harbor Press) ]; solid phase direct labeling of the RIA using the I-125 marker [ see Morel et al (1988) mol.Immunol.25(1):7) ]; solid phase direct Biotin-avidin EIA [ Cheung et al (1990) Virology176:546 ]; and directly labeled RIA [ Moldenhauer et al (1990) Scand. J. Immunol.32:77 ]. Typically, such assays involve the use of a purified antigen bound to a solid surface or cells having one of an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to a solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more.

Other techniques include, for example, epitope mapping methods, such as x-ray analysis of crystals of antigen-antibody complexes, which provide atomic resolution of the epitope. Other methods monitor binding of antibodies to antigen fragments or mutant variants of the antigen, where loss of binding due to modification of amino acid residues within the antigen sequence is often taken as an indicator of epitope composition. Furthermore, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate a particular short peptide from a combinatorial phage display peptide library. The peptides were then considered as leads for the determination of epitopes for antibodies used to screen peptide libraries. For epitope mapping, computational algorithms have also been developed that have been shown to map topographically discontinuous epitopes.

As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like.

Aspects of the disclosure are described in further detail in the following sections.

The present disclosure relates to a pharmaceutical combination comprising components (a) and (B) as defined herein, wherein the pharmaceutical combination is in the form of a combined preparation for simultaneous, separate or sequential use. Component (a) relates to an anti-BST 1 antibody as defined herein. Component (B) relates to a cytidine analog or a pharmaceutically acceptable salt thereof.

anti-BST 1 antibodies

The antibodies of the pharmaceutical combination of the invention are characterized by specific functional characteristics or properties of the antibodies. For example, antibodies specifically bind to human BST 1. Preferably, the antibodies of the invention have high affinityE.g. at 8x10^-7M or less, even more typically 1x10^-8K of M or less_DIn combination with BST 1. The anti-BST 1 antibodies preferably exhibit one or more of the following characteristics, wherein the antibodies exhibit two specific uses:

with an EC of 50nM or less, 10nM or less, 1nM or less, 100pM or less, or more preferably 10pM or less₅₀Binding to human BST 1;

binding to human cells expressing BST 1.

In one embodiment, the antibody preferably binds to an epitope present in BST1, which epitope is not present in other proteins. Preferably, the antibody does not bind to the protein of interest, e.g., the antibody does not substantially bind to other cell adhesion molecules. In one embodiment, the antibody may be internalized into cells expressing BST 1. Standard assays for assessing antibody internalization are known in the art and include, for example, MabZap or HumZap internalization assays.

Standard assays for assessing the binding ability of antibodies to BST1 can be performed at the protein level or the cellular level and are known in the art and include, for example, ELISA, Western blot, RIA, and the like,

Assays and flow cytometry analyses. Suitable assays are detailed in the examples. It can also be tested by standard tests known in the art, such as by

Systematic analysis to assess the binding kinetics (e.g., binding affinity) of the antibody. To assess binding to Raji or Daudi b cell tumor cells, Raji cells (ATCC accession No. CCL-86) or Daudi cells (ATCC accession No. CCL-213) are obtained from publicly available sources such as the American Type Culture Collection and used in standard assays such as flow cytometry analysis.

Monoclonal antibodies

Preferred antibodies of the pharmaceutical combination of the invention are the monoclonal antibody BST1_ a2 and variants thereof isolated and structurally characterized as described in examples 1-4.V of BST1_ A2_HThe amino acid sequence is shown in SEQ ID NO 2. V of BST1_ A2_KThe amino acid sequence is shown in SEQ ID NO 4.

Since the antibody can bind to BST1, V_HAnd V_KThe sequences may be varied to generate other anti-BST 1 binding molecules. The BST1 binding of the variant antibodies can be tested using the binding assays (e.g., ELISA) described above and in the examples.

Thus, in one aspect, the pharmaceutical combination comprises an antibody comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 2 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 4, wherein the antibody specifically binds to BST1, preferably human BST 1.

The CDR regions of the antibodies disclosed herein are depicted using the Kabat system [ Kabat, E.A. et al (1991) protein Sequences of Immunological Interest (Sequences of Proteins of Immunological Interest), fifth edition, U.S. Department of Health and public service (US Department of Health and Human Services), NIH publication No. 91-3242 ].

The ability of an antibody to bind to BST1 can be measured using the binding assays described above and in the examples (e.g., ELISA, a,

Analysis) of the test.

In another preferred embodiment, the antibody has:

comprises the heavy chain variable region CDR1 of SEQ ID NO. 10;

comprises the c heavy chain variable region CDR2 of SEQ ID NO 12 or SEQ ID NO 51;

comprises the heavy chain variable region CDR3 of SEQ ID NO. 14;

comprises the light chain variable region CDR1 of SEQ ID NO. 16;

18, comprising the light chain variable region CDR2 of SEQ ID NO; and

comprises the light chain variable region CDR3 of SEQ ID NO. 20.

It is well known in the art that the CDR3 domain, independently of the CDR1 and/or CDR2 domain, can determine the binding specificity of an antibody to the corresponding antigen individually and that a number of antibodies with the same binding specificity can be generated predictably based on the common CDR3 sequence see, for example, Klimka et al (2000) British J.of Cancer 83(2) 252. 260 (describing the generation of humanized anti-CD 30 antibodies using only the heavy chain variable domain CDR3 of the murine anti-CD 30 antibody Ki-4), Beiboer et al (2000) J.mol. biol.296:833-849 (describing the recombinant epithelial-2 (EGP-2) antibody using only the heavy chain CDR3 sequence of the parent murine MOC-31 anti-EGP-2 antibody), Rad et al (1998) Proc. Acad. Sci.95: 10H-H.

Accordingly, the present invention provides a pharmaceutical combination comprising a monoclonal antibody comprising one or more heavy and/or light chain CDR3 domains, said CDR3 domain being derived from an antibody derived from a human or non-human animal, wherein the monoclonal antibody is capable of specifically binding to BST 1. In certain aspects, a monoclonal antibody comprises one or more heavy and/or light chain CDR3 domains from a nonhuman antibody, such as a mouse or rat antibody, wherein the monoclonal antibody is capable of specifically binding to BST 1. Within some embodiments, such inventive antibodies comprising one or more heavy and/or light chain CDR3 domains from a non-human antibody (a) are capable of competing for binding with a corresponding parent non-human antibody; (b) retaining the functional characteristics of the corresponding parent non-human antibody; (c) binds to the same epitope as the corresponding parent non-human antibody; and/or (d) has similar binding affinity to a corresponding parent non-human antibody.

Within other aspects, the invention provides pharmaceutical combinations comprising a monoclonal antibody comprising one or more heavy and/or light chain CDR3 domains, said CDR3 domain being from a human antibody, e.g., a human antibody obtained from a non-human animal, wherein the human antibody is capable of specifically binding to BST 1. Within other aspects, a monoclonal antibody comprises one or more heavy and/or light chain CDR3 domains, the CDR3 domain being from a first human antibody, such as a human antibody obtained from a non-human animal, wherein the first human antibody is capable of specifically binding to BST1, and wherein a CDR3 domain from the first human antibody replaces a CDR3 domain in the human antibody lacking binding specificity for BST1 to produce a second human antibody capable of specifically binding to BST 1. Within some embodiments, such inventive antibodies comprising one or more heavy and/or light chain CDR3 domains from a first human antibody (a) are capable of competing for binding with a corresponding parent first human antibody; (b) retaining the functional characteristics of the corresponding parent first human antibody; (c) binds to the same epitope as the corresponding parent first human antibody; and/or (d) has a similar binding affinity as the corresponding parent first human antibody.

Antibodies with specific germline sequences

In certain embodiments of the invention, the antibody comprises a heavy chain variable region from a particular germline heavy chain immunoglobulin gene and/or a light chain variable region from a particular germline light chain immunoglobulin gene.

For example, in a preferred embodiment, the invention provides a pharmaceutical combination comprising an isolated monoclonal antibody, or antigen binding portion thereof, contained as murine V_H1-39 Gene, murine V_H1-80 Gene or murine V_H69-1 Gene product or derived from murine V_H1-39 Gene, murine V_H1-80 Gene or murine V_HThe heavy chain variable region of the 69-1 gene, wherein the antibody specifically binds to BST 1. In a further preferred embodiment, the invention is embodied in a carrierPharmaceutical combinations are provided comprising an isolated monoclonal antibody, or antigen binding portion thereof, as embodied by murine V_K4-55 Gene, murine V_K4-74 Gene or murine V_K44-1 Gene products or derived from murine V_K4-55 Gene, murine V_K4-74 Gene or murine V_K44-1 gene, wherein the antibody specifically binds to BST 1.

In yet another preferred embodiment, the present invention provides a pharmaceutical combination comprising an isolated monoclonal antibody, or antigen binding portion thereof, wherein the antibody:

comprising as murine V_H1-39 gene (the gene contains the nucleotide sequence shown in SEQ ID NO:35 and 36) or derived from mouse V_H1-39 gene; comprising as murine V_K4-55 gene (the gene contains the nucleotide sequence shown in SEQ ID NO:40, 41 and 42) or derived from mouse V_K4-55 gene; and specifically binds to BST1, preferably human BST 1. Having a V_H1-39 and V_KAn example of an antibody of the 4-55 gene (with sequences described above) is BST1_ a 2.

In a further preferred embodiment, the present invention provides a pharmaceutical combination comprising an isolated monoclonal antibody, or antigen binding portion thereof, wherein the antibody:

comprising as murine V_H1-80 gene (the gene contains the nucleotide sequence shown in SEQ ID NO:33 and 34) or derived from mouse V_H1-80 gene; comprising as murine V_K4-74 gene (the gene includes the nucleotide sequence shown in SEQ ID NO:37, 38 and 39) or mouse V-derived gene_K4-74 gene; and specifically binds to BST1, preferably human BST 1. Having a V_H1-80 and V_KAn example of an antibody for the 4-74 gene (with sequences described above) is BST1_ a 1.

In a further preferred embodiment, the present invention provides a pharmaceutical combination comprising an isolated monoclonal antibody, or antigen binding portion thereof, wherein the antibody:

comprising as murine V_H69-1 gene (the gene includes SEQ ID NO: 1)Nucleotide sequences shown in IDNOs 68 and 69) or derived from murine V_HThe heavy chain variable region of the 69-1 gene; comprising as murine V_K44-1 gene (the gene includes the nucleotide sequence shown in SEQ ID NO:70, 71 and 72) or derived from mouse V_KThe light chain variable region of the 44-1 gene; and specifically binds to BST1, preferably human BST 1. Having a V_HAnd V_KAn example of an antibody for a gene (having the sequence described above) is BST1_ A3.

As used herein, an antibody comprises a heavy or light chain variable region that is "the product of" or "derived from" a particular germline sequence if the variable region of the antibody is obtained from a system using murine germline immunoglobulin genes. Such systems include screening of phage-displayed murine immunoglobulin gene libraries with the antigen of interest. Antibodies that are "products" of "or" derived from "murine germline immunoglobulin sequences can thus be identified by: the nucleotide or amino acid sequence of the antibody is compared to the nucleotide or amino acid sequence of a murine germline immunoglobulin and the murine germline immunoglobulin sequence that is closest in sequence (i.e., most identical%) to the sequence of the antibody is selected. An antibody that is "the product of" or "derived from" a particular murine germline immunoglobulin sequence may contain amino acid differences compared to that germline sequence, such as those resulting from naturally occurring somatic mutations or artificially introduced site-directed mutations. However, the selected antibody is typically at least 90% identical in amino acid sequence to the amino acid sequence encoded by a murine germline immunoglobulin gene and contains amino acid residues that identify the antibody as murine when compared to germline immunoglobulin amino acid sequences from other species (e.g., human germline sequences). In certain instances, the antibody may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, antibodies derived from a particular murine germline sequence exhibit no more than 10 amino acid differences compared to the amino acid sequence encoded by the murine germline immunoglobulin gene. In certain instances, an antibody may exhibit no more than 5, or even no more than 4,3, 2, or 1 amino acid differences compared to the amino acid sequence encoded by the germline immunoglobulin gene.

Homologous antibodies

In yet another embodiment of the invention, the antibody comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of a preferred antibody described herein (e.g., BST1_ a2), and wherein the antibody retains the desired functional properties of the parent anti-BST 1 antibody.

For example, the present invention provides a pharmaceutical combination comprising an isolated monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID No. 2; the light chain variable region comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO. 4; and the antibody binds to human BST 1. Such antibodies may have an EC of 50nM or less, 10nM or less, 1nM or less, 100pM or less, or more preferably 10pM or less₅₀Binding to human BST 1.

The antibodies also bound to CHO cells transfected with human BST 1.

In various embodiments, for example, the antibody can be a human antibody, a humanized antibody, or a chimeric antibody.

In other embodiments, V_HAnd/or V_KThe amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth above. V having a sequence similar to that shown above can be obtained in the following manner_HAnd V_KV of the same zone height (i.e., 80% or more)_HAnd V_KAntibodies to the regions: nucleic acid molecules encoding SEQ ID NOS: 6 and 8 are mutagenized (e.g., site-directed or PCR-mediated mutagenesis) and the encoded altered antibodies are subsequently tested for retained function using the functional assays described herein.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e.,% homology is the number of identical positions/total number of positions x 100), taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm as described in the non-limiting examples below.

The percent identity between two amino acid sequences can be determined using the PAM120 weighted residue table, gap length penalty of 12, and gap penalty of 4 using the E.Meyers and W.Miller algorithms [ Compout.Appl.biosci. (1988)4:11-17], which have been incorporated into the ALIGN program (version 2.0). Furthermore, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch [ J.mol.biol. (1970)48:444-453] algorithm, which has been incorporated into the GCG software package (available from http:// www.gcg.com) GAP program, using either the Blossum 62 matrix or the PAM250 matrix, and a GAP weight of 16, 14,12, 10, 8, 6, or 4, and a length weight of 1,2, 3,4, 5, or 6.

Additionally or alternatively, the protein sequences of the invention may further be used as "query sequences" to search against public databases, e.g., to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al (1990) J.mol.biol.215: 403-10. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the antibody molecules of the invention. For comparison purposes, gapped BLAST can be used as described in Altschul et al (1997) Nucleic Acids Res.25(17):3389-3402, in order to obtain gapped alignments (). When utilizing BLAST and gapped BLAST programs, the default parameters for each program (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

Antibodies with conservative modifications

In certain embodiments, the antibodies of the invention comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences; and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences comprises a designated amino acid sequence based on the preferred antibody described herein (i.e., BST1_ a2), or a conservative modification thereof, and wherein the antibody retains the desired functional properties of an anti-BST 1 antibody. Accordingly, the present invention provides a pharmaceutical combination comprising an isolated monoclonal antibody, or antigen binding portion thereof,the isolated monoclonal antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences; and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein: the heavy chain variable region CDR3 sequence comprises the amino acid sequence of SEQ ID No. 14 and conservative modifications thereof; the light chain variable region CDR3 sequence comprises the amino acid sequence of SEQ ID NO:20 and conservative modifications thereof; and the antibody has an EC of 50nM or less, 10nM or less, 1nM or less, 100pM or less, or more preferably 10pM or less₅₀Binding to human BST 1.

The antibodies also bound to CHO cells transfected with human BST 1.

In a preferred embodiment, the heavy chain variable region CDR2 sequence comprises the amino acid sequence of SEQ ID NOs 12 or 51 and conservative modifications thereof; and the light chain variable region CDR2 sequence comprises the amino acid sequence of SEQ ID NO. 18 and conservative modifications thereof. In another preferred embodiment, the heavy chain variable region CDR1 sequence comprises the amino acid sequence of SEQ ID No. 10 and conservative modifications thereof; and the light chain variable region CDR1 sequence comprises the amino acid sequence of SEQ ID NO 16 and conservative modifications thereof. In another preferred embodiment, the heavy chain variable region CDR3 sequence comprises the amino acid sequence of SEQ ID NO. 14 and conservative modifications thereof; and the light chain variable region CDR3 sequence comprises the amino acid sequence of SEQ ID NO. 20 and conservative modifications thereof.

In various embodiments, the antibody can be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

The present invention relates to antibodies and more particularly to methods for the detection of antibodies that bind to a protein or protein, such as a protein or protein, using the methods described herein, and to methods for the detection of such proteins and/or proteins using such antibodies and/or proteins, and more particularly to methods for the detection of such proteins and/or proteins using such antibodies and/or proteins and/or polypeptides and/or proteins and/or polypeptides and/or.

The heavy chain CDR1 sequence of SEQ ID No. 10 can comprise one or more conservative sequence modifications, such as one, two, three, four, five or more amino acid substitutions, additions or deletions; light chain CDR1 sequence SEQ ID NO 16 may comprise one or more conservative sequence modifications, such as one, two, three, four, five or more amino acid substitutions, additions or deletions; the heavy chain CDR2 sequence shown in SEQ ID NO 12 or 51 may comprise one or more conservative sequence modifications, such as one, two, three, four, five or more amino acid substitutions, additions or deletions; the light chain CDR2 sequence shown in SEQ ID No. 18 can comprise one or more conservative sequence modifications, such as one, two, three, four, five or more amino acid substitutions, additions or deletions; the heavy chain CDR3 sequence shown in SEQ ID No. 14 can comprise one or more conservative sequence modifications, such as one, two, three, four, five or more amino acid substitutions, additions or deletions; and/or the light chain CDR3 sequence shown in SEQ ID No. 20 can comprise one or more conservative sequence modifications, such as one, two, three, four, five or more amino acid substitutions, additions or deletions.

Antibodies that bind to the same epitope as the anti-BST 1 antibodies of the invention

In another embodiment, the invention provides a pharmaceutical combination comprising an antibody that binds to the same epitope on human BST1 as BST1_ a2 (i.e., an antibody that has the ability to cross-compete with BST1_ a2 for binding to BST 1).

Such cross-competitive antibodies can be identified based on their ability to cross-compete with BST1_ a2 in a standard BST1 binding assay. For example, BIAcore analysis, ELISA assays, or flow cytometry can be used to demonstrate cross-competition with BST1_ a 2. The ability of the test antibody to inhibit the binding of BST1_ a2 to human BST1 indicates that the test antibody can compete with BST1_ a2 for binding to human BST1 and thus bind to the same epitope on human BST1_ a 2.

Engineered and modified antibodies

Can be used with one or more of the V's disclosed herein_HAnd/or V_LAntibodies of sequence (which can be used as starting materials to engineer modified antibodies) to make the antibodies disclosed herein, the modified antibodies can have altered properties compared to the starting antibodies. By modifying one or both variable regions (i.e., V)_HAnd/or V_L)Antibodies are engineered, for example, with one or more amino acids within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, antibodies can be engineered by modifying residues within the constant region (e.g., to alter the effector function of the antibody).

In certain embodiments, CDR grafting can be used to engineer the variable regions of antibodies. Antibodies interact with a target antigen primarily through amino acid residues located in the six heavy and light chain Complementarity Determining Regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than sequences outside the CDRs. Because the CDR sequences are responsible for the majority of the antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a naturally occurring particular antibody by constructing expression vectors that include CDR sequences from that naturally occurring particular antibody grafted onto framework sequences from different antibodies with different properties (see, e.g., Riechmann, L. et al (1998) Nature332: 323-.

Therefore, resistThe antibody may contain V of monoclonal antibody BST1_ A2_HAnd V_KThe CDR sequences, in turn, may contain framework sequences different from those of the antibody.

Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA Sequences for murine heavy and light chain variable region genes can be found in the IMGT (international imminogenetics) murine germline sequence database (available under hypertext transfer protocol// www.imgt.cines.fr/; the contents of each are expressly incorporated herein by reference. As another example, germline DNA sequences for murine heavy and light chain variable region genes can be found in the Genbank database.

The antibody protein sequences were compared against the compiled protein sequence database using one of the sequence similarity search methods known to those skilled in the art as BLAST for gaps [ Altschul et al (1997) Nucleic Acids Research 25:3389-3402 ]. BLAST is a heuristic algorithm in which statistically significant alignments between antibody sequences and database sequences may contain highly scored segment pairs (HSPs) of the aligned fields. Fragments whose scores cannot be increased by expansion or pruning are referred to as hits (hit). Briefly, the nucleotide sequences in the database were translated and the region between FR1 to FR3 framework regions and comprising FR1 to FR3 framework regions was retained. The database sequences had an average length of 98 residues. Repeat sequences that match exactly over the entire length of the protein are removed. BLAST searches for proteins were performed using the program blastp, default standard parameters (except for low complexity filters, which were closed) and the substitution matrix BLOSUM62, the filter for the first 5 hits that produced sequence matches. The nucleotide sequence was translated in all six frames and the frames with no stop codon in the database sequence-matched segment were considered potential hits. This was in turn confirmed using the BLAST program tblastx, which translated the antibody sequences in all six frames and compared these translations to nucleotide sequences dynamically translated in all six frames in the database.

Identity is the exact amino acid match between an antibody sequence and a protein database over the entire sequence length. Positive (identity + substitution match) is not identical, but there are amino acid substitutions as directed by the BLOSUM62 substitution matrix. If the antibody sequences match the two database sequences with the same identity, then the hit with the most positive is determined to be the matching sequence hit.

Preferred framework sequences for use in the antibodies disclosed herein are those that are structurally similar to the framework sequences used in the selected antibodies of the invention, e.g., V, used with the preferred monoclonal antibodies of the invention_H1-80 framework sequence, V_H1-39 framework sequence, V_K4-74 framework sequences and/or V_K4-55 framework sequences are similar. Can make V_HCDR1, CDR2 and CDR3 sequences and V_KThe CDR1, CDR2, and CDR3 sequences are grafted onto framework regions having sequences identical to those present in the germline immunoglobulin gene from which the framework sequences were generated, or the CDR sequences may be grafted onto framework regions containing one or more mutations compared to the germline sequence. For example, it has been found that in certain instances, mutating residues within the framework regions is beneficial to maintain or enhance the antigen binding ability of the antibody (see, e.g., U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al).

Another type of variable region modification is to modify V_HAnd/or V_KAmino acid residues within the CDR1 region, CDR2 region, and/or CDR3 region are mutated to improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations, and the effect on antibody binding or other functional property of interest can be evaluated in vitro or in vivo assays as described and provided in the examples herein. In some embodiments, conservative modifications are introduced (as discussed above). Alternatively, non-conservative modifications may be made. The mutation may be an amino acid substitution, addition or deletion, but is preferably a substitution. In addition, typically no more than one, two, three, four or five residues within a CDR region are altered, but as will be appreciated by those skilled in the artThe variants within his region (e.g., framework region) may be larger.

Engineered antibodies of the invention include, for example, antibodies to V for improved antibody properties_HAnd/or V_KAntibody modified with internal framework residues. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "back mutate" one or more framework residues into the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody was produced. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody was raised.

Another type of framework modification involves mutating one or more residues within the framework regions, or even within one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also referred to as "deimmunization" and is described in more detail in U.S. patent publication No. 2003/0153043.

In addition to or as an alternative to modifications within the framework or CDR regions, antibodies can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In addition, the antibody may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation to again alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

In one embodiment, modification C _H1, thereby altering (e.g., increasing or decreasing) the number of cysteine residues in the hinge region. This process is further described in U.S. Pat. No. 5,677,425. Change C _H1, to, for example, facilitate assembly of the light and heavy chains or to increase or decrease stability of the antibody.

In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. Furniture setIn particular, one or more amino acid mutations are introduced into the C of the Fc-hinge fragment_H2-C _H3 domain interface region, such that the antibody has impaired staphylococcal protein a (SpA) binding relative to native Fc-hinge domain SpA binding. This method is described in further detail in U.S. Pat. No. 6,165,745.

In another embodiment, the antibody is modified to increase its biological half-life. A variety of approaches are possible. For example, one or more of the following mutations may be introduced: T252L, T254S, T256F, as described in U.S. patent No. 6,277,375. Alternatively, to increase biological half-life, one may use C _H1 or C_LIntraregionally altered antibodies to contain a salvage receptor (salvagerecepter) binding epitope taken from two loops of the CH2 domain of the IgG Fc region as described in U.S. patent nos. 5,869,046 and 6,121,022.

In another embodiment, the antibody is produced as a single antibody (Unibody) as described in WO2007/059782, which is incorporated herein by reference in its entirety.

In other embodiments, the Fc region is altered by substituting at least one amino acid residue for a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 may be substituted with a different amino acid residue, thereby conferring altered affinity of the antibody for an effector ligand, but retaining the antigen binding ability of the parent antibody. The affinity-altered effector ligand may be, for example, an Fc receptor or the C1 component of complement. This method is described in further detail in U.S. Pat. nos. 5,624,821 and 5,648,260.

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be substituted with a different amino acid residue, thereby conferring altered C1q binding and/or reduced or eliminated Complement Dependent Cytotoxicity (CDC) to the antibody. This method is described in further detail in U.S. Pat. No. 6,194,551.

In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to alter the ability of the antibody to fix complement. This method is further described in PCT publication No. WO 94/29351.

In yet another example, the amino acid sequence is modified by modifying one or more of the amino acids at the following positions: 238. 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 to modify the Fc region to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for Fc γ receptors. The method is further described in PCT publication No. WO 00/42072 to Presta. In addition, the binding sites for Fc γ R1, Fc γ RII, Fc γ RIII and FcRn on human IgG1 have been mapped and variants with improved binding have been described (see Shield, R.L. et al (2001) J.biol.chem.276: 6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 have been shown to improve binding to Fc γ RIII. In addition, the following combinatorial mutants showed improved Fc γ RIII binding: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A. Other ADCC variants are described, for example, in WO 2006/019447.

In yet another example, the Fc region is modified to increase the half-life of the antibody, typically by increasing binding to the FcRn receptor, as described in, for example, PCT/US2008/088053, US 7,371,826, US 7,670,600, and WO 97/34631. In another embodiment, the antibody is modified to increase its biological half-life. A variety of approaches are possible. For example, one or more of the following mutations may be introduced: T252L, T254S, T256F, as described in U.S. patent No. 6,277,375 to Ward. Alternatively, to increase biological half-life, one may use C _H1 or C_LIntraregionally altering the antibody to include a salvage receptor binding epitope taken from C of the Fc region of IgG _H2, as described in U.S. Pat. nos. 5,869,046 and 6,121,022.

In yet another embodiment, the glycosylation of the antibody is modified. For example, aglycosylated antibodies (i.e., antibodies lacking glycosylation) can be produced. For example, glycosylation can be altered to increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for the antigen. Such a method is described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 to Co et al, and may be accomplished by removing asparagine at location 297.

Additionally or alternatively, antibodies can be produced that have altered glycosylation patterns, such as low fucosylated antibodies with a reduced number of fucosyl residues or antibodies with an increase in the bisecting GlcNac structure. It is sometimes referred to in the art as "engineered glycoform". Such altered glycosylation patterns have been shown to increase the ADCC capacity of the antibody. Such carbohydrate modifications can generally be accomplished in two ways; for example, in some embodiments, the antibody is expressed in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells for expression of recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. Reference to

For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8(α (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates, the Ms704, Ms705, and Ms709FUT8 were generated by targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors^-/-Cell lines [ see U.S. patent publication No. 2004/0110704, U.S. patent No. 7,517,670, and Yamane-Ohnuki et al (2004) Biotechnol. Bioeng.87:614-22]. As another example, EP 1,176,195 to Hanai et al describes F with functional disruptionCell lines of the UT8 gene encoding fucosyltransferases such that antibodies expressed in such cell lines exhibit low fucosylation by reducing or eliminating α 1,6 bond-associated enzymes Hanai et al also describe cell lines with or without low enzymatic activity for adding fucose to N-acetylglucosamine bound to the Fc region of antibodies, e.g., the rat myeloma cell line YB2/0(ATCC CRL 1662). alternatively, engineered glycoforms, particularly afucosylation, can be prepared using small molecule inhibitors of glycosylation pathway enzymes [ see, e.g., Rothman et al (1989) mol. Immunol.26(12): 113-1123; Elbein (1991) FASEB J.5: 3055; PCT/US 2009/042610; and US patent No. 7,700,321]. PCT publication No. WO 03/035835 describes a variant CHO cell line Lec13 cell that has a reduced ability to link fucose to an Asn (297) -linked carbohydrate, which also results in low fucosylation of antibodies expressed in the host cell [ see also Shields, R.L. et al (2002) J.biol.chem.277:26733-26740]PCT publication No. WO 99/54342 describes cell lines engineered to express a glycoprotein-modified glycosyltransferase (e.g., β (1,4) -N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit an increase in the bipartite GlcNac structure, which results in an increase in antibody ADCC activity [ see also Umana et al (1999) nat. Biotech.17:176-]。

For example, fucosidase α -L-fucosidase removes fucosyl residues of antibodies [ Tarentino, A.L. et al (1975) biochem.14:5516-23 ].

Another modification of the antibodies described herein that is encompassed by the present invention is pegylation. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as an active ester or aldehyde derivative of PEG, under the following conditions: in which one or more PEG groups are attached to the antibody or antibody fragment. Preferably, pegylation is performed via an acylation reaction or alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy-or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and are applicable to the antibodies of the present invention. See, for example, EP 0154316 and EP 0401384.

In further embodiments, the antibody may comprise a label. Reference to "labeled" herein means that the compound has attached at least one element, isotope, or chemical compound. In general, markers fall into three categories: a) an isotopic label, which can be a radioisotope or a heavy isotope; b) magnetic, electrical, thermal; and c) a coloured or luminescent dye; although labels also include enzymes and particles, such as magnetic particles. Preferred labels include, but are not limited to, fluorescent lanthanide complexes (europium and terbium containing complexes); and fluorescent labels include, but are not limited to, quantum dots, fluorescein, rhodamine (rhodamine), tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene (stilbene), fluorescein (Lucifer Yellow), karst Blue (Cascade Blue), Texas Red (Texas Red), Alexa dyes, Cy dyes, and other labels described in Richard p.

Joint

The present invention provides pharmaceutical combinations comprising an antibody-partner conjugate in which the antibody is linked to a partner (partner) via a chemical linker. In some embodiments, the linker is a peptide-based linker; other linkers include hydrazine linkers and disulfide linkers. In addition to linker and partner attachment, the present invention also provides cleavable linker arms that are suitable for use in attaching substantially any molecular species. The linker arm aspect of the invention is exemplified herein by reference to the connection of the linker arm of the invention to a therapeutic moiety. However, it will be apparent to those skilled in the art that the linker may be attached to a wide variety of substances, including, but not limited to, diagnostic agents, analytical agents, biomolecules, targeting agents, detectable labels, and the like.

The use of peptide-based and other linkers in antibody-partner conjugates is described in U.S. provisional patent application serial No. 60/295,196, 60/295,259, 60/295342, 60/304,908, 60/572,667, 60/661,174, 60/669,871, 60/720,499, 60/730,804 and 60/735,657 and U.S. patent application serial No. 10/160,972, 10/161,234, 11/134,685, 11/134,826 and 11/398,854 and U.S. patent No. 6,989,452 and PCT patent application No. PCT/US2006/37793, all of which are incorporated herein by reference. Other linkers are described in U.S. patent nos. 6,214,345; U.S. patent application No. 2003/0096743; and U.S. patent application No. 2003/0130189; de Groot et al, j.med.chem.42,5277 (1999); de Groot et al, j.org.chem.43,3093 (2000); de Groot et al, j.med.chem.66,8815, (2001); WO 02/083180; carl et al, J.Med.chem.Lett.24,479, (1981); dubowchik et al, Bioorg & Med. chem. Lett.8,3347 (1998); and U.S. provisional patent application No. 60/891,028.

In one aspect, the invention relates to linkers useful for linking targeting groups to therapeutic agents and markers. In another aspect, the invention provides linkers that confer stability to a compound, reduce its toxicity in vivo, or otherwise favorably affect its pharmacokinetics, bioavailability, and/or pharmacodynamics. It is generally preferred that in such embodiments, once the drug is delivered to its site of action, the linker is cleaved, releasing the active drug. Thus, in one embodiment, the linker is traceless, such that once removed from the therapeutic agent or marker (such as during activation), no trace of the presence of the linker is left. In another embodiment, the linker is characterized by its ability to cleave at a site in or near the target cell (such as at a site of therapeutic action or marker activity). Such cleavage may be enzymatic in nature. This feature helps to reduce systemic activation of the therapeutic agent or marker, reducing toxicity and systemic side effects. Preferred cleavable groups for enzymatic cleavage include peptide bonds, ester linkages, and disulfide linkages. In other embodiments, the linker is pH sensitive and can be cleaved by a change in pH.

One aspect of the present invention is the ability to control the speed of splice cutting. A rapidly cutting splice is often required. However, in some embodiments, a more slowly cutting linker is preferred. For example, in sustained release formulations or in formulations with fast and slow release components, it may be useful to provide a linker that cuts more slowly. WO 02/096910 provides several specific ligand-drug complexes with hydrazine linkers. However, there is no way to "tune" the linker composition depending on the desired rate of cyclization, and the particular compounds described cleave the ligand from the drug at a rate that is slower than the preferred rate for many drug-linker conjugates. In contrast, hydrazine linkers can provide a range of cyclization rates (from extremely fast to extremely slow), thus allowing the selection of a particular hydrazine linker based on the desired cyclization rate.

For example, very fast cyclization can be achieved with hydrazine linkers that yield a single 5-membered ring upon cleavage. A preferred cyclization rate for targeted delivery of cytotoxic agents to cells is achieved using hydrazine linkers that upon cleavage yield two 5-or a single 6-membered rings (which result from a linker with two methyl groups at geminal position). The gem-dimethyl (gem-dimethyl) effect has been shown to accelerate the rate of cyclization reactions compared to a single 6-membered ring that does not have two methyl groups at the gem positions. This is due to the stress being relieved in the ring. However, substituents can sometimes slow the reaction rather than speed it up. The cause of retardation is often attributable to steric hindrance. For example, with gem-carbon CH₂Geminal dimethyl substitution allows for a faster cyclization reaction to occur than when it is used.

However, it is important to note that in some embodiments, a more slowly cutting linker may be preferred. For example, in sustained release formulations or in formulations with fast and slow release components, linkers that provide slower cleavage may be useful. In certain embodiments, slow cyclization rates are achieved using hydrazine linkers that yield a single 6-membered ring (absent geminal dimethyl substitution) or a single 7-membered ring upon cleavage. The linker also serves to stabilize the therapeutic agent or marker from degradation during circulation. This feature provides significant benefits, as such stabilization results in an extended circulating half-life of the linked agent or marker. Linkers are also used to reduce the activity of the linked agent or marker, thereby rendering the conjugate relatively benign in circulation and having the desired effect, e.g., toxicity, upon activation at the desired site of action. For therapeutic agent conjugates, this feature of the linker is used to improve the therapeutic index of the agent.

The stabilizing group is preferably selected to limit the clearance and metabolism of the therapeutic agent or marker by enzymes that may be present in the blood or non-target tissue, and further selected to limit the transport of the agent or marker into the cell. The stabilizing group serves to block degradation of the agent or marker and may also serve to provide other physical characteristics of the agent or marker. The stabilizing group may also improve the stability of the agent or marker during storage in formulated or non-formulated form.

Ideally, a stabilizing group can be used to stabilize a therapeutic agent or marker if it protects the agent or marker from degradation when tested by storing the agent or marker in human blood for 2 hours at 37 ℃ and results in less than 20%, preferably less than 10%, more preferably less than 5% and even more preferably less than 2% of the agent or marker being cleaved by enzymes present in human blood under the given test conditions. The invention also relates to conjugates containing these linkers. More specifically, the invention relates to the use of prodrugs useful in the treatment of diseases, particularly in cancer chemotherapy. In particular, the use of linkers described herein provides prodrugs that exhibit high specificity of action, reduced toxicity, and improved stability in blood relative to structurally similar prodrugs. The linkers of the invention as described herein may be present at multiple positions within the partner molecule.

Thus, a linker is provided that may contain any of a variety of groups as part of its chain that will cleave at an enhanced rate in vivo (e.g., in the bloodstream) relative to a construct lacking such groups. Conjugates of the linker arm with therapeutic and diagnostic agents are also provided. The linker may be used to form prodrug analogs of the therapeutic agent, and may be used to reversibly attach the therapeutic or diagnostic agent to a targeting agent, detectable label, or solid support. The linker may be incorporated into a complex comprising a cytotoxin.

The attachment of the prodrug to the antibody may yield additional safety advantages over conventional antibody conjugates of cytotoxic drugs. Activation of the prodrug can be achieved by esterases in tumor cells and in several normal tissues, including plasma. The levels of relevant esterase activity in humans have been shown to be very similar to those observed in rats and non-human primates, albeit at a level lower than that observed in mice. Activation of the prodrug can also be achieved by cleavage by glucuronidase. In addition to cleavable peptide, hydrazine or disulfide groups, one or more self-immolative linker groups are optionally introduced between the cytotoxin and the targeting agent. These linker groups can also be described as spacer groups and contain at least two reactive functional groups. Typically, one chemical functionality of the spacer group is bonded to a chemical functionality of the therapeutic agent (e.g., a cytotoxin) and the other chemical functionality of the spacer group is used to bond to a chemical functionality of the targeting agent or cleavable linker. Examples of chemical functional groups of the spacer group include hydroxyl, thiol, carbonyl, carboxyl, amino, keto, and thiol.

The self-immolative linker is typically a substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heteroalkyl. In one embodiment, the alkyl or aryl group may contain 1 to 20 carbon atoms. They may also contain polyethylene glycol moieties.

Exemplary spacer groups include, for example, 6-aminohexanol, 6-mercaptohexanol, 10-hydroxydecanoic acid, glycine and other amino acids, 1, 6-hexanediol, β -alanine, 2-aminoethanol, cysteamine (2-aminoethanethiol), 5-aminopentanoic acid, 6-aminocaproic acid, 3-maleimidobenzoic acid, tetrachlorophthalide, α -substituted tetrachlorophthalides, carbonyl groups, animal esters, nucleic acids, peptides, and the like.

The spacer may be used to introduce additional molecular mass and chemical functionality into the cytotoxin-targeting agent complex. Generally, the additional mass and functional groups will affect the serum half-life and other properties of the complex. Thus, by careful selection of spacer groups, cytotoxic complexes with a range of serum half-lives can be generated.

When a plurality of spacers are present, the same or different spacers may be used.

Additional linker moieties may be used which preferably impart increased solubility or decreased aggregation properties to the conjugate utilizing a linker containing such moiety, or which modulate the rate of hydrolysis of the conjugate, such linkers not necessarily being self-immolative. In one embodiment, the linking moiety is a substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroalkyl, or unsubstituted heteroalkyl, each of which may be linear, branched, or cyclic. For example, the substitution may be lower (C)₁-C₆) Alkyl, alkoxy, alkylthio, alkylamino or dialkylamino. In certain embodiments, the linker comprises an acyclic moiety. In another embodiment, the linker comprises any positively or negatively charged amino acid polymer, such as polylysine or polyarginine. The linker may comprise a polymer, such as a polyethylene glycol moiety. Additionally, the linker may comprise, for example, a polymeric component and a small chemical moiety. In a preferred embodiment, such linkers comprise a polyethylene glycol (PEG) moiety.

The PEG moiety may have a length between 1 and 50 units. Preferably, the PEG has 1-12 repeating units, more preferably 3-12 repeating units, more preferably 2-6 repeating units, or even more preferably 3-5 repeating units, and optimally 4 repeating units. The linker may consist entirely of PEG moieties, or it may also contain additional substituted or unsubstituted alkyl or heteroalkyl groups. It is useful to incorporate PEG as part of this moiety to enhance the water solubility of the complex. In addition, the PEG moiety reduces the extent of aggregation that may occur during drug-antibody conjugation.

For a further discussion of the type of cytotoxin, linkers, and other methods for conjugating therapeutic agents to antibodies, see also PCT publication No. WO 2007/059404 to Gangwar et al entitled "Cytotoxic Compounds and conjugates" (cytoxic Compounds and conjugates); saito, G, et al, (2003) adv. drug Deliv. Rev.55: 199-215; trail, P.A. et al (2003) Cancer Immunol.Immunother.52: 328-337; payne, G. (2003) Cancer Cell 3: 207-; allen, T.M. (2002) nat. Rev. cancer2: 750-; pastan, I, and Kreitman, R.J, (2002) curr, Opin, Investig, drugs3: 1089-; senter, P.D. and Springer, CJ. (2001) adv. Drag Deliv. Rev.53:247-264, each of which is incorporated by reference in its entirety.

Partner molecule

The drug combination may include an antibody conjugated to a partner molecule such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radiotoxin. Such conjugates are also referred to herein as "immunoconjugates". Immunoconjugates comprising one or more cytotoxins are referred to as "immunotoxins". A cytotoxin or cytotoxic agent includes any agent that is harmful (e.g., killing) to a cell.

Examples of the disclosed partner molecules include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin (daunorubicin), dihydroxy anthrax dione, mitoxantrone, mithramycin (mithramycin), actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Examples of partner molecules also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (daunorubicin), and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin (bleomycin), mithramycin, and antrocin (ammycin) (AMC), and antimitotics (e.g., vincristine and vinblastine).

Other preferred examples of partner molecules that can be conjugated to preferred antibodies include duocarmycin (duocarmycin), calicheamicin (calicheamicin), maytansine (maytansine) and auristatin (auristatin) and derivatives thereof. Examples of calicheamicin antibody conjugates are commercially available (

American home Products, inc (american home Products)).

Preferred examples of partner molecules are CC-1065 and duocarmycin. CC-1065 was first isolated in 1981 by the company Upjohn from Streptomyces zelissis (Hanka et al, J.Antibiot.31:1211 (1978); Martin et al, J.Antibiot.33:902 (1980); Martin et al, J.Antibiot.34:1119(1981)) and was found to have potent antitumor and antimicrobial activity both in vitro and in experimental animals (Li et al, Cancer Res.42:999 (1982)). CC-1065 binds to double-stranded B-DNA in the minor groove (minor groove) (Swenson et al, Cancer Res.42:2821(1982)), sequence preferences are 5' -d (A/GNTTA) -3' and 5' -d (AAAAA) -3', and the N3 position of 3' -adenine is alkylated by its CPI left-hand unit present in the molecule (Hurley et al, Science 226:843 (1984)).

Despite its potent and broad antitumor activity, CC-1065 cannot be used in humans because it causes late death in experimental animals. Many analogs and derivatives of CC-1065 and duocarmycin are known in the art. A review of the structure, synthesis and properties of many of these compounds has been made. See, e.g., Boger et al, Angew.chem.int.Ed.Engl.35:1438 (1996); and Boger et al, chem.Rev.97:787 (1997). A group of Kyowa Hakko Kogya Co., Ltd, has prepared many CC-1065 derivatives. See, e.g., U.S. Pat. nos. 5,101,038, 5,641,780, 5,187,186, 5,070,092, 5,703,080, 5,070,092, 5,641,780, 5,101,038, and 5,084,468; and published PCT application WO 96/10405 and published european application 0537575 a 1. Derivatives of CC-1065 were also actively prepared by Upjohn, Pharmacia Upjohn. See, for example, U.S. patent nos. 5,739,350, 4,978,757, 5,332,837 and 4,912,227.

Physical Properties of antibodies

The antibodies disclosed herein can be further characterized by a variety of physical properties of the anti-BST 1 antibodies. Based on these physical properties, various assays can be used to examine and/or distinguish between different classes of antibodies.

In some embodiments, an antibody disclosed herein may contain one or more glycosylation sites in the light chain or heavy chain variable region. The presence of one or more glycosylation sites in the variable region can lead to increased immunogenicity of the antibody or to altered pK of the antibody (due to altered antigen binding) [ Marshall et al (1972) Annu Rev Biochem 41: 673-702; gala FA and Morrison SL (2004) J Immunol 172: 5489-94; wallick et al (1988) J Exp Med 168: 1099-109; spiro RG (2002) Glycobiology12: 43R-56R; parekh et al (1985) Nature 316: 452-7; mimura et al (2000) Mol Immunol37:697-706 ]. Glycosylation is known to occur at motifs containing N-X-S/T sequences. Variable region glycosylation can be tested using the glycoimprinting assay (glycoimprinting assay) which cleaves antibodies to produce fabs, and then using an assay that measures periodate oxidation and Schiff base (Schiff base) formation. Alternatively, variable region glycosylation can be tested using Dionex light chromatography (Dionex-LC), which cleaves carbohydrates from Fab into monosaccharides and analyzes the content of individual carbohydrates. In some cases, it is preferred to have an anti-BST 1 antibody that does not contain variable region glycosylation. This can be achieved by selecting antibodies that do not contain a glycosylation motif in the variable region or by mutating residues within the glycosylation motif using standard techniques well known in the art.

In a preferred embodiment, the antibodies disclosed herein do not contain asparagine isomerization sites. Deamidation or isoaspartic acid action may occur on the N-G or D-G sequences, respectively. Deamidation or isoaspartic acid action results in the production of isoaspartic acid, which reduces the stability of the antibody by creating a kinked structure at the carboxyl end of the side chain rather than outside the backbone. Isoaspartic acid production can be measured using an isoyield assay that uses reverse phase HPLC to test isoaspartic acid.

Each antibody has a unique isoelectric point (pI), but the antibody will typically fall within a pH range between 6 and 9.5. The pI of the IgG1 antibody typically falls within a pH range of 7-9.5 and the pI of the IgG4 antibody typically falls within a pH range of 6-8. Antibodies can have a pI outside this range. While the role is generally unknown, there is a conjecture: antibodies with pI outside the normal range may have some unfolding and instability under in vivo conditions. Isoelectric points can be tested using a capillary isoelectric focusing assay that produces a pH gradient and can be focused using a laser to increase accuracy [ Janni et al (2002) Electrophoresis 23: 1605-11; ma et al (2001) Chromatographia 53: S75-89; hunt et al (1998) J chromanogr A800: 355-67 ]. In some cases, it is preferred to have an anti-BST 1 antibody with a pI value that falls within the normal range. This can be achieved by selecting antibodies with pI in the normal range or by mutating charged surface residues using standard techniques well known in the art.

Each antibody will have a melting temperature indicative of thermostability [ Krishnhamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71]. Higher thermostability indicates greater overall antibody stability in vivo. The melting point of antibodies can be measured using a variety of techniques such as differential scanning calorimetry [ Chen et al (2003) Pharm Res 20: 1952-60; ghirland et al (1999) Immunol Lett 68:47-52]。T_M1Indicating the temperature at which the antibody initially unfolds. T is_M2Indicating the temperature at which the antibody completes unfolding. Generally, T of the antibodies disclosed herein is preferred_M1Greater than 60 ℃, preferably greater than 65 ℃, even more preferably greater than 70 ℃. Alternatively, the thermal stability of antibodies can be measured using circular dichroism [ Murray et al (2002) J. chromatogr Sci 40:343-9]。

In a preferred embodiment, antibodies are selected that do not degrade rapidly. Fragmentation of anti-BST 1 antibodies can be measured using Capillary Electrophoresis (CE) and MALDI-MS as is well known in the art [ Alexander AJ and Hughes DE (1995) anal. chem.67:3626-32 ].

In another preferred embodiment, the antibody is selected to have minimal aggregation effects. Aggregation may result in the triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties. Generally, antibodies with 25% or less aggregation, preferably 20% or less, even more preferably 15% or less, even more preferably 10% or less, and even more preferably 5% or less, are acceptable. Aggregation can be measured by a variety of techniques well known in the art including Size Exclusion Column (SEC) High Performance Liquid Chromatography (HPLC) and light scattering to identify monomers, dimers, trimers, or multimers.

Methods of engineering antibodies

As discussed above, with V as disclosed herein_HAnd V_Kanti-BST 1 antibodies of sequence may be used by modifying V_HAnd/or V_KThe sequence or constant region linked thereto, to generate novel anti-BST 1 antibodies. Thus, the structural features of the preferred anti-BST 1 antibodies (e.g., BST1_ a2) are used to generate structurally related anti-BST 1 antibodies that retain at least one functional property of the preferred antibodies, such as binding to human BST 1. For example, one or more CDR regions of BST1_ a2, or mutations thereof, can be recombined with known framework regions and/or other CDRs to produce other recombinantly engineered anti-BST 1 antibodies, as discussed above. Other types of modifications include those described in the previous section. Starting materials for the engineering process are one or more of the V provided herein_HAnd/or V_KA sequence or one or more CDR regions thereof. To generate engineered antibodies, it is not necessary to actually prepare (i.e., express as a protein) a peptide having one or more of the V provided herein_HAnd/or V_KAn antibody having the sequence or one or more CDR regions thereof. Rather, the information contained in the sequence is used as starting material to generate a "second generation" sequence derived from the original sequence, and the "second generation" sequence is then prepared and expressed as a protein.

Standard molecular biology techniques can be used to prepare and express altered antibody sequences.

Preferably, the antibody encoded by the altered antibody sequence is an antibody that retains one, some, or all of the functional properties of the anti-BST 1 antibodies described herein, including but not limited toLimited to: (a) at 1 × 10^-7M or less binds to human BST 1; (b) binding to human CHO cells transfected with BST 1.

The functional properties of the altered antibody can be assessed using standard assays available in the art and/or described herein, such as those described in the examples (e.g., flow cytometry, binding assays).

Mutations can be introduced randomly or selectively along all or part of the anti-BST 1 antibody coding sequence, and the resulting modified anti-BST 1 antibodies can be screened for binding activity and/or other functional properties as described herein. Methods of mutagenesis have been described in the art. For example, PCT publication No. WO 02/092780 describes methods of generating and screening for antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, PCT publication No. WO 03/074679 describes a method of optimizing the physicochemical properties of an antibody using a computational screening method.

Nucleic acid molecules encoding antibodies

Also disclosed are nucleic acid molecules encoding the antibodies disclosed herein. The nucleic acid may be present in whole cells, in cell lysates, or in partially purified or substantially pure form. Nucleic acids are "isolated" or "become substantially pure" when they are separated and purified from other cellular components or other impurities (e.g., other cellular nucleic acids or proteins) by standard techniques, including alkali/SDS treatment, CsCl banding, tube chromatography, agarose gel electrophoresis, and other techniques well known in the art. See, e.g., Ausubel et al (1987) molecular biology Protocols, Green Press and Wiley Interscience Press, New York. For example, these nucleic acids may be DNA or RNA and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

Nucleic acids encoding the antibodies disclosed herein can be obtained using standard molecular biology techniques. For antibodies expressed by a hybridoma, the cdnas encoding the light and heavy chains of the antibody produced by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), nucleic acids encoding the antibodies can be recovered from the library.

Preferred nucleic acid molecules are those encoding the BST1_ A2 monoclonal antibody V_HAnd V_KThose of the sequence. Encoding of V for BST1_ A2_HThe DNA sequence of the sequence is shown in SEQ ID NO 6. Encoding of V for BST1_ A2_KThe DNA sequence of the sequence is shown in SEQ ID NO 8.

Other preferred nucleic acids are those having at least 80% sequence identity, such as at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to one of the sequences set forth in SEQ ID NOS 6 and 8, which encode a preferred antibody or antigen-binding portion thereof.

The percent identity between two nucleic acid sequences is the number of positions in the sequence where the nucleotides are identical, and takes into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Sequence comparison and percent identity determination between two sequences can be accomplished using mathematical algorithms, such as the Meyers and Miller algorithms described above or the xtast program of Altschul.

In addition, preferred nucleic acids of the invention comprise one or more CDR-encoding portions of the nucleic acid sequences shown in SEQ ID Nos. 6 and 8. In this embodiment, the nucleic acid may encode the heavy and light chain CDR1, CDR2 and/or CDR3 sequences of BST1_ a 2.

With SEQ ID NOS 6 and 8 (V)_HAnd V_KSequences) nucleic acids having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to such CDR-encoding portions are also preferred nucleic acids of the invention. Such nucleic acids may differ from the corresponding portions of SEQ ID NO 6 and 8 in the non-CDR-encoding regions and/or in the CDR-encoding regions. Where the difference is in a CDR encoding region, the CDR regions of the nucleic acid encoded by the nucleic acid typically contain one or more conservative sequence modifications as defined herein, as compared to the corresponding CDR sequences of BST1_ a 2.

Once the code V is obtained_HAnd V_KSegmented DNA fragments which can be further manipulated by standard recombinant DNA techniques, e.g., to map the variable regionsThe gene is converted into a full-length antibody chain gene, into a Fab fragment gene, or into a scFv gene. In these manipulations, V will be encoded_KOr V_HIs operably linked to another DNA segment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used in this context means that two DNA segments are linked such that the amino acid sequences encoded by the two DNA segments remain in frame.

Code V_HThe DNA of the region can be isolated by subjecting the coding V_HDNA of (3) and a DNA encoding a heavy chain constant region (C)_H1、C _H2 and C_H3) Operably linked to convert to a full-length heavy chain gene. The sequence of the murine constant region gene is known in the art [ see, for example, Kabat, E.A. et al (1991) for immunologically significant protein Sequences (Sequences of Immunological Interest), fifth edition, U.S. department of health and public service, NIH publication No. 91-3242]And DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG1 or IgG4 constant region. For Fab fragment heavy chain genes, the gene encoding V_HDNA of (1) and encoding only heavy chain C _H1 constant region is operably linked to another DNA molecule.

Code V_L/V_KThe DNA of the region can be isolated by subjecting the coding V_LDNA of (1) and encoding light chain constant region C_LOperably linked to convert to a full-length light chain gene (as well as a Fab light chain gene). The sequence of the murine light chain constant region gene is known in the art [ see, e.g., Kabat, E.A. et al (1991) for immunologically significant protein Sequences (Sequences of proteins of Immunological Interest), fifth edition, U.S. department of health and public service, NIH publication No. 91-3242]And DNA fragments containing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region can be a kappa or lambda constant region.

For the production of scFv genes, encoding VH and encoding V_L/V_KThe DNA fragment of (1) and a linker encoding a flexible linker (e.g., encoding an amino acid sequence (Gly4-Ser)₃) To another one ofThe segments are operably linked such that V_HAnd V_L/V_KThe sequence may be expressed as having V's connected by a flexible linker_L/V_KAnd V_HContinuous single-stranded proteins of the region [ see, e.g., Bird et al (1988) Science 242: 423-426; huston et al (1988) Proc.Natl.Acad.Sci.USA 85: 5879-; McCafferty et al (1990) Nature348:552-554]。

Production of monoclonal antibodies

BST1, or a fragment or derivative thereof, can be used as an immunogen to generate antibodies that immunospecifically bind to the immunogen. The immunogen may be isolated by any conventional means. One skilled in the art will appreciate that a number of methods can be used to generate Antibodies, for example as described in antibody Laboratory Manual (Antibodies, A Laboratory Manual), Harlow and DavidLane, Cold Spring Harbor Press (Cold Spring Harbor Laboratory) (1988), Cold Spring Harbor, N.Y.. It will also be appreciated by those skilled in the art that binding fragments or Fab fragments of the mock antibodies can also be prepared from genetic information by various methods [ [ Practical methods of Antibody Engineering: A Practical Approach (Borreboeck, ed., C.), 1995, Oxford University Press (Oxford University Press), Oxford; J.Immunol.149,3914-3920 (1992).

Antibodies can be raised against specific domains of BST 1. Hydrophilic fragments of BST1 can be used as immunogens for antibody production.

In generating antibodies, screening for the desired antibody can be accomplished by techniques known in the art, such as ELISA (enzyme-linked immunosorbent assay). For example, to select antibodies that recognize a particular domain of BST1, the resulting hybridomas can be tested for products that bind to a BST1 fragment containing such a domain. To select an antibody that specifically binds to the first BST1 homolog but does not specifically bind (or binds with less affinity to) the second BST1 homolog, the selection can be based on a positive binding to the first BST1 homolog and an absence of binding (or a reduction in binding to) the second BST1 homolog. Similarly, to select antibodies that specifically bind BST1 but do not specifically bind (or bind with less affinity) to different isoforms of the same protein (such as different glycoforms having the same core peptide as BST1), selection can be based on positive binding to BST1 and the absence of binding (or reduction of binding thereto) to different isoforms (e.g., different glycoforms).

Thus, antibodies (such as monoclonal antibodies) are disclosed that bind to BST1 with greater affinity (e.g., at least 2-fold, such as at least 5-fold, especially at least 10-fold greater affinity) than different isoforms (e.g., glycoforms) of BST 1.

Polyclonal antibodies as used herein are a heterogeneous population of antibody molecules derived from the serum of an immunized animal. Unfractionated immune serum can also be used. Various methods known in the art can be used to generate polyclonal antibodies against BST1, fragments of BST1, BST 1-related polypeptides, or fragments of BST 1-related polypeptides. For example, one way is to purify the polypeptide of interest or synthesize the polypeptide of interest using, for example, solid phase peptide synthesis methods well known in the art. See, e.g., the guidelines for Protein Purification (Guide to Protein Purification), eds, Murray p.deutcher, meth.enzymol. vol. 182 (1990); solid Phase Peptide Synthesis (Solid Phase Peptide Synthesis), eds. Greg b.fields, meth.enzymol. Vol.289 (1997); kiso et al, chem.pharm.Bull. (Tokyo)38:1192-99, 1990; mostafavi et al, biomed.Pept.Proteins Nucleic Acids 1:255-60, 1995; fujiwara et al, chem.Pharm.Bull. (Tokyo)44: 1326-. The selected polypeptide can then be used to immunize various host animals including, but not limited to, rabbits, mice, rats, etc., by injection to produce polyclonal or monoclonal antibodies. Depending on the host species, various adjuvants (i.e., immunostimulants) may be used to enhance the immune response, including but not limited to Freund's complete or incomplete adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin (keyhole limpet hemocyanin), dinitrophenol, and adjuvants such as BCG (bacillus Calmette-Guerin) or Corynebacterium parvum (Corynebacterium parvum). Other adjuvants are also well known in the art.

To prepare monoclonal antibodies (mabs) against BST1, any technique for producing antibody molecules by continuous cell lines in culture can be used. For example, the hybridoma technology originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma (trioma) technology, the human B-cell hybridoma technology [ Kozbor et al (1983) Immunology Today4:72] and the EBV-hybridoma technology producing human Monoclonal Antibodies [ Cole et al (1985), Monoclonal Antibodies and Cancer Therapy (Monoclonal Antibodies and Cancer Therapy), Alan R.Liss GmbH., pp.77-96 ]. The antibodies may belong to any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. Hybridomas producing monoclonal antibodies can be cultured in vitro or in vivo. Monoclonal antibodies can be produced in sterile animals using known techniques (PCT/US90/02545, incorporated herein by reference).

The preferred animal system for preparing hybridomas is the murine system. The generation of hybridomas in mice is a well established method. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion methods are also known.

Monoclonal antibodies include, but are not limited to, human monoclonal antibodies and chimeric monoclonal antibodies (e.g., human-mouse chimeras).

Chimeric or humanized antibodies can be prepared based on the sequence of the non-human monoclonal antibody prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from a non-human hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to generate chimeric antibodies, murine variable regions can be linked to human constant regions using methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al). To generate humanized antibodies, murine CDR regions can be inserted into a human framework using methods known in the art (see, e.g., Winter U.S. patent No. 5,225,539 and Queen et al U.S. patent nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370).

Fully human antibodies can be produced using transgenic or transchromosomal mice, wherein the mice are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but canHuman heavy and light chain genes are expressed. The transgenic mice are immunized in the normal manner with a selected antigen (e.g., all or part of BST 1). Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma techniques. The large human immunoglobulin transgene carried by the transgenic mice rearranges during B cell differentiation and subsequently undergoes class switching and somatic mutation. Thus, using this technique, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. These transgenic and transchromosomal mice include HuMAb

(

Company Limited) and KM

Mice of the strain. HuMAb

Strain (A)

Gmbh) are described in Lonberg and huskzar (1995, int. rev. immunol.13: 65-93). For a detailed discussion of such techniques for producing human antibodies and human monoclonal antibodies and large protocols for producing such antibodies, see, e.g., U.S. Pat. No. 5,625,126, U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,661,016, and U.S. Pat. No. 5,545,806. KM (Kernel) matrix

Strains refer to mice carrying a human heavy chain transgene and a human light chain transchromosome, and are described in detail in PCT publication No. WO 02/43478 to Ishida et al.

In addition, other transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to produce the large anti-BST 1 antibodies of the invention. For example, other transgenic systems known as Xenomouse (Amgen, Inc.); such mice are described, for example, in U.S. Pat. Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963 to Kucherlapati et al.

Fully human antibodies recognizing selected epitopes can be generated using a technique known as "guided selection". In this method, selected non-human monoclonal antibodies (e.g., mouse antibodies) are used to guide the selection of fully human antibodies recognizing the same epitope [ Jespers et al (1994) Biotechnology 12:899-903 ].

In addition, other transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used to generate anti-BST 1 antibodies. For example, a mouse known as a "TC mouse" carrying a human heavy chain transchromosome and a human light chain transchromosome; such mice are described in Tomizuka et al (2000) Proc. Natl. Acad. Sci. USA 97: 722-. In addition, cattle carrying human heavy and light chain transfectants have been described in the art [ Kuroiwa et al (2002) Nature Biotechnology 20:889-894 and PCT publication No. WO2002/092812 ] and can be used to generate anti-BST 1 antibodies.

The human monoclonal antibodies of the invention can also be prepared using SCID mice in which human immune cells have been reconstituted so that a human antibody response can be generated following immunization. Such mice are described, for example, in U.S. patent nos. 5,476,994 and 3,698,767.

The antibodies disclosed herein can be produced by: phage display technology is used to generate and screen polypeptide libraries for binding to selected targets [ see, e.g., Cwirla et al, Proc. Natl. Acad. Sci. USA 87,6378-82, 1990; devlin et al, Science 249,404-6, 1990; scott and Smith, Science 249,386-88, 1990; and Ladner et al, U.S. patent No. 5,571,698 ]. The basic concept of the phage display method is to establish a physical association between the DNA encoding the polypeptide to be screened and the polypeptide. This physical association is provided by the phage particle which displays the polypeptide as part of a capsid surrounding the phage genome encoding the polypeptide. Establishing a physical association between a polypeptide and its genetic material allows for the simultaneous large-scale screening of a very large number of phages carrying different polypeptides. Phage displaying a polypeptide with affinity for a target bind to the target, and the phage are enriched by affinity screening for the target. The identity of the polypeptides displayed by these phage can be determined from their corresponding genomes. Using these methods, polypeptides identified as having binding affinity for a desired target can then be synthesized in large quantities by conventional means. See, for example, U.S. patent No. 6,057,098, which is hereby incorporated by reference in its entirety, including all tables, figures, and claims. In particular, such phage can be used to display antigen binding domains expressed by a reservoir or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds an antigen of interest can be selected or identified using the antigen (e.g., using a labeled antigen or an antigen bound to or captured to a solid surface or bead). The phage used in these methods are typically filamentous phage comprising fd and M13 binding domains expressed from phage and Fab, Fv, or disulfide-stabilized Fv antibody domains recombinantly fused to phage gene III or gene VIII proteins. Phage display methods that can be used to produce the antibodies of the invention include those described in Brinkman et al (1995) J.Immunol.methods 182: 41-50; ames et al (1995) J.Immunol.methods 184: 177-186; kettleborough et al, Eur.J.Immunol.24: 952-; persic et al (1997) Gene 1879-18; burton et al (1994) Advances in Immunology 57: 191-280; PCT application No. PCT/GB 91/01134; PCT publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO92/18619, WO 93/11236, WO 95/15982, WO 95/20401; and those disclosed in U.S. Pat. nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108; each of which is incorporated by reference in its entirety.

As described in the above references, after phage selection, the antibody coding regions can be isolated from the phage and used to produce whole antibodies (including human antibodies) or any other desired antigen binding fragments and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described in detail below. For example, methods known in the art may also be used, such as those described in PCT publication No. WO 92/22324; mullinax et al (1992) BioTechniques 12(6) 864-869; and Sawai et al (1995) AJRI 34: 26-34; and Better et al (1988) Science 240: 1041-.

Examples of techniques that can be used to produce single chain Fv's and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; huston et al (1991), Methods in Enzymology 203: 46-88; shu et al (1993) PNAS 90: 7995-7999; and those described in Skerra et al (1988) Science 240: 1038-.

The present invention provides functionally active fragments, derivatives or analogues of anti-BST 1 immunoglobulin molecules. By functionally active it is meant that the fragment, derivative or analogue is capable of eliciting an anti-idiotypic antibody (i.e. tertiary antibody) which recognizes the same antigen as an antibody derived from the fragment, derivative or analogue recognizes. In particular, in a particular embodiment, the antigenicity of the idiotype of an immunoglobulin molecule can be enhanced by deleting the framework and CDR sequences that specifically recognize the C-terminus of the CDR sequences of the antigen. To determine which CDR sequences bind to the antigen, synthetic peptides containing CDR sequences can be used in the binding assay, as well as the antigen, by any binding assay method known in the art.

Also disclosed are antibody fragments, such as but not limited to F (ab')₂Fragments and Fab fragments. Antibody fragments that recognize a particular epitope can be generated by known techniques. The F (ab')2 fragment consists of a variable region, a light chain constant region and a heavy chain-large C _H1 domain and is produced by pepsin digestion of the antibody molecule. Fab fragments by reduction of F (ab')₂Disulfide bonds of the fragments. Also disclosed are heavy and light chain dimers of the antibodies disclosed herein, or any minimal fragment thereof, such as Fv or Single Chain Antibodies (SCAs) [ e.g., as described in U.S. patent nos. 4,946,778; bird, (1988) Science 242: 423-42; huston et al (1988) Proc. Natl. Acad. Sci. USA85: 5879-; and Ward et al (1989) Nature 334:544-5]Or any other molecule having the same specificity as an antibody of the invention. The heavy and light chain fragments of the Fv region are joined by an amino acid bridge to produce a single chain polypeptide, which forms a single chain antibody. Techniques for assembling functional Fv fragments in E.coli [ Skerra et al (1988) Science 242:1038-]。

Also disclosed are fusion proteins of the immunoglobulins (or functionally active fragments thereof) disclosed herein, for example wherein the immunoglobulin is fused at the N-terminus or C-terminus via a covalent bond (e.g. a peptide bond) to an amino acid sequence of another protein (or portion thereof, preferably a portion of at least 10, 20 or 50 amino acids of the protein) which is not larger than the immunoglobulin. Preferably, the immunoglobulin or fragment thereof is covalently linked to another protein at the N-terminus of the constant domain. As described above, such fusion proteins can facilitate purification, increase in vivo half-life, and facilitate antigen delivery across the epithelial barrier to the immune system.

The immunoglobulins disclosed herein include modified analogs and derivatives, i.e., modified by covalent attachment of any type of molecule, so long as such covalent attachment does not impair immunospecific binding. For example, but not by way of limitation, derivatives and analogs of immunoglobulins include derivatives and analogs that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, protease cleavage, linkage to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the like. In addition, the analog or derivative may contain one or more non-canonical amino acids.

Immunization of mice

Mice can be immunized with purified or enriched preparations of BST1 antigen and/or recombinant BST1 or cells expressing BST 1. Preferably, the mice are 6-16 weeks old when first infused. For example, purified or recombinant preparations (100 μ g) of the BST1 antigen can be used to immunize mice intraperitoneally.

Cumulative experience with various antigens has shown that mice respond when immunized Intraperitoneally (IP) with antigen in freund's complete adjuvant. However, adjuvants other than Freund's adjuvant have been found to be effective. In addition, intact cells were found to be highly immunogenic in the absence of adjuvant. The immune response can be monitored during the course of an immunization protocol, and plasma samples obtained by retro-orbital bleeding. Plasma can be screened by ELISA (as described below) to test for satisfactory titers. Mice can be boosted intravenously with antigen for 3 consecutive days, sacrificed 5 days later and spleens removed. In one embodiment, an A/J mouse strain (Jackson Laboratories, Burmese, Balport) may be used.

Transfectomas producing monoclonal antibodies

Antibodies disclosed herein can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods, as is well known in the art [ e.g., Morrison, S. (1985) Science229:1202 ].

For example, to express an antibody or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., live PCR amplification using cDNA clones expressing an antibody hybridoma of interest), and the DNA can be inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" means that the antibody gene is linked into a vector such that transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used.

The host cell may be co-transfected with two expression vectors, a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide. Both vectors may contain the same selectable marker that enables equivalent expression of the heavy and light chain polypeptides. Alternatively, a single vector encoding both the heavy and light chain polypeptides may be used. In such cases, the light chain should be placed before the heavy chain to avoid excessive toxic free heavy chain [ Proudfoot (1986) Nature 322: 52; kohler (1980) Proc. Natl. Acad. Sci. USA 77:2197 ]. The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

The antibody gene can be inserted into an expression vector by standard methods (e.g., linking the antibody gene fragment to complementary restriction sites on the vector, or blunt-ended if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein can be used to generate full-length antibody genes of any antibody isotype by: it is inserted into an expression vector which already encodes the heavy chain constant region and the light chain constant region of the desired isotype, so that V_HC in sections and carriers_HThe segments are operably connected and V_KC in sections and carriers_LThe segments are operably connected. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a protein other than an immunoglobulin).

The term "regulatory sequence" is intended to include promoters, enhancers and other Expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology, Methods in Enzymology 185, Academic Press, Adademic Press, san Diego, Calif. (1990)) the skilled artisan will appreciate that the design of Expression vectors, including the selection of regulatory sequences, can be determined depending on factors such as the selection of the host cell to be transformed, the level of Expression of the desired protein, etc. preferred regulatory sequences for Expression in mammalian host cells include viral elements that direct the Expression of high levels of protein in mammalian cells, such as promoters derived from Cytomegalovirus (CMV), monkey 40 (40), viruses (e.g., major promoters (SV P) and late viral promoters (MLP) and late viral promoters such as late viral promoters from adenovirus viruses, such as the SV promoter, SV 6335. Biobema promoter, SV promoter from simian adenovirus promoter, SV 83. Sn-368. further, Skyphoma virus promoter from Biobema virus, Sn et al, Skyo, Skol 2. Skov, Skov. the Expression vector can be used in the same.

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors disclosed herein may also carry other sequences, such as sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all of Axel et al). For example, selectable marker genes typically confer resistance to drugs (such as G418, hygromycin or methotrexate) on host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (used in conjunction with methotrexate selection/amplification in DHFR-host cells) and the neo gene (for G418 selection).

To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The term "transfection" of various forms is intended to include the commonly used to prokaryotic or eukaryotic host cell into exogenous DNA large variety of techniques, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection. While it is theoretically possible to express the antibodies disclosed herein in prokaryotic or eukaryotic host cells, it is preferred to express the antibodies in eukaryotic cells, and most preferably mammalian host cells, because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a correctly folded and immunologically active antibody. Prokaryotic expression of the gene of the lead antibody has been reported to be ineffective for the production of high yields of active antibody [ Boss, M.A. and Wood, C.R. (1985) Immunology Today 6:12-13 ].

Preferred mammalian host cells for expression of the recombinant antibodies disclosed herein include Chinese Hamster Ovary (CHO) cells, along with vectors such as the major intermediate early Gene promoter elements from human cytomegalovirus [ fooking et al, 1986, Gene 45: 101; the DHFR-CHO cells described in Urlaut et al (1990) Biotechnology 8:2], Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-. In particular, another preferred expression system for use with NSO myeloma cells is the GS gene expression system disclosed in WO87/04462(Wilson), WO 89/01036(Bebbington), and EP 338,841 (Bebbington).

Various host expression vector systems can be used to express the antibody molecules disclosed herein. The host-expression system represents a vector by which the coding sequence of interest can be produced and subsequently purified, but also represents a cell that can express the antibody molecules disclosed herein in situ when transformed or transfected with the appropriate nucleotide coding sequence. Including, but not limited to, microorganisms such as bacteria (e.g., escherichia coli, bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces (Saccharomyces), Pichia (Pichia)) transformed with a recombinant yeast expression vector containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant viral expression vectors containing antibody coding sequences (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors large enough to contain antibody coding sequences (e.g., Ti plasmid); or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a variety of expression vectors may be advantageously selected depending on the intended use of the expressed antibody molecule. For example, when the protein is intended to be produced in large quantities, e.g., for the production of pharmaceutical compositions comprising antibody molecules, vectors directing high levels of expression of fusion protein products that are easy to purify may be required. Such vectors include, but are not limited to, the E.coli expression vector pUR278(Ruther et al (1983) EMBO J.2:1791) in which the antibody coding sequence can be ligated into the vector separately in frame with the lac Z coding region, thereby producing a fusion protein; pIN vectors [ Inouye and Inouye (1985) Nucleic acids sRs.13: 3101-3109; van Heeke and Schuster (1989) J.biol.chem.24:5503-5509 ]; and similar pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to the matrix glutathione-agarose beads, followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) was used as a vector for expression of foreign genes. The virus grows in Spodoptera frugiperda (Spodoptera frugiperda) cells. Antibody coding sequences can be individually cloned into non-essential regions of the virus (e.g., polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., polyhedrin promoter). In mammalian host cells, a variety of viral-based expression systems (e.g., adenoviral expression systems) are available.

As discussed above, host cell lines can be selected that modulate the expression of the inserted sequences or modify and process the gene products in a particular manner as desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product may be important to the function of the protein.

For long-term, high-yield production of recombinant antibodies, stable expression is preferred. For example, a cell line stably expressing an antibody of interest can be generated by: cells are transfected with an expression vector comprising the nucleotide sequence of the antibody and a nucleotide sequence of a selectable marker (e.g., neomycin or hygromycin) and selected for expression of the selectable marker. Such engineered cell lines are particularly useful for screening and evaluating compounds that interact directly or indirectly with antibody molecules.

The expression level of antibody molecules can be increased by vector amplification [ for review, see Bebbington and Hentschel, use of gene amplification-based vectors in DNA cloning for expressing cloned genes in mammalian cells (use of vector-based gene amplification for the expression of cloned genes in DNA cloning), Vol.3 (academic Press, New York, 1987) ]. When the marker in the vector system expressing the antibody is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with an antibody gene, antibody production will also increase [ Crouse et al, 1983, mol.cell.biol.3:257 ].

When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, an antibody is produced by: culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell or, more preferably, to allow the antibody to be secreted into the medium in which the host cell is grown. After recombinant expression of the antibody molecule, it may be purified by any method known in the art for purifying antibody molecules, for example by chromatography (e.g., ion exchange chromatography, affinity chromatography such as with protein a or a specific antigen, and size column chromatography), centrifugation, differential solubilization, or by any other standard technique for purifying proteins.

Alternatively, any fusion protein can be easily purified by using an antibody specific to the expressed fusion protein. For example, the system described by Janknecht et al allows easy purification of undenatured fusion proteins expressed in human cell lines [ Janknecht et al, 1991, Proc. Natl. Acad. Sci. USA 88:8972-]. In this system, the gene of interest is subcloned into a vaccinia virus recombinant plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as the matrix binding domain of the fusion protein. Loading Ni with extracts from recombinant vaccinia virus infected cells²⁺On a azaacetic acid-agarose column and selective with imidazole-containing bufferThe histidine-tagged protein was eluted.

Characterization of antibodies binding to antigens

Antibodies produced by these methods can then be selected by: the affinity and specificity with the purified polypeptide of interest is first screened and, if necessary, the results are compared to the affinity and specificity of the antibody and the polypeptide that is desired to be excluded from binding. Binding of the antibody to BST1 can be tested by, for example, standard ELISA. The screening method may comprise immobilizing the purified polypeptide in individual wells of a microtiter plate. The solution containing the potential antibody or group of antibodies is then placed in individual microtiter wells and incubated for about 30 minutes to 2 hours. The microtiter wells are then washed, and a labeled secondary antibody (e.g., an anti-mouse antibody conjugated to alkaline phosphatase if the antibody produced is a mouse antibody) is added to each well and incubated for about 30 minutes, followed by washing. A substrate is added to each well and when antibodies to the immobilized polypeptide are present, a color reaction will occur.

The antibodies so identified can then be further analyzed for affinity and specificity in selected assay designs. In developing immunoassays against target proteins, the purified target proteins serve as standards with which to judge the sensitivity and specificity of immunoassays using selected antibodies. Because the binding affinities of various antibodies may differ, certain antibody pairs (e.g., in a sandwich assay) may spatially interfere with each other, etc., the analytical potency of an antibody may be a more important measure than the absolute affinity and specificity of an antibody.

One skilled in the art will recognize that many methods may be employed in generating antibodies or binding fragments and screening and selecting for the affinity and specificity of various polypeptides, but such methods do not alter the scope of the present invention.

To determine whether the selected anti-BST 1 monoclonal antibodies bind to a unique epitope, each antibody can be biotinylated using commercially available reagents (Pierce, rockford, il). Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using BST1 coated ELISA plates. The binding of biotinylated mAb can be detected using a streptavidin-alkaline phosphatase probe.

To determine the isotype of the purified antibody, an isotype ELISA can be performed using reagents specific for the particular isotype antibody.

The reactivity of anti-BST 1 antibodies to BST1 antigen can be further tested by Western blotting. Briefly, BST1 can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens were transferred to nitrocellulose membranes, blocked with 10% fetal bovine serum, and probed with the monoclonal antibodies to be tested.

The binding specificity of the antibodies disclosed herein can also be determined by monitoring (e.g., by flow cytometry) the binding of the antibody to cells expressing BST 1. Typically, cell lines, such as CHO cell lines, may be transfected with an expression vector encoding BST 1. The transfected protein may comprise a tag (such as a myc-tag), preferably at the N-terminus, for detection using an antibody directed against the tag. Binding of the antibody to BST1 can be determined by incubating the transfected cells with the antibody and detecting the bound antibody. Binding of the antibody to the tag on the transfected protein can be used as a positive control.

The specificity of antibodies against BST1 can be further studied by: using the same method used to determine binding to BST1, it was determined whether the antibody binds to other proteins (such as another member of the Eph family).

Immunoconjugates

The drug combination may comprise an anti-BST 1 antibody or fragment thereof conjugated to a therapeutic moiety such as a cytotoxin, drug (e.g., immunosuppressant), or radiotoxin. Such conjugates are referred to herein as "immunoconjugates". Immunoconjugates comprising one or more cytotoxins are referred to as "immunotoxins". A cytotoxic or cytotoxic agent includes any agent that is detrimental (e.g., kills) cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin (daunorubicin), dihydroxyanthrax dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunorubicin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin (bleomycin), mithramycin and Antromycin (AMC)), and antimitotics (e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can be conjugated to antibodies in a pharmaceutical combination include duocarmycin (duocarmycin), calicheamicin (calicheamicin), maytansine (maytansine), and auristatin (auristatin), and derivatives thereof. Examples of calicheamicin antibody conjugates are commercially available (

American Home Products, inc (American Home Products)).

Cytotoxins may be conjugated to antibodies using linker technology available in the art. Examples of the types of linkers that have been used to couple cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfide bonds, and peptide-containing linkers. Linkers can be selected that are susceptible to cleavage, for example, at low pH within the lysosomal compartment or to cleavage by proteases, such as proteases preferentially expressed in tumor tissue, such as cathepsins (e.g., cathepsin B, C, D).

Examples of cytotoxins are described, for example, in U.S. patent nos. 6,989,452, 7,087,600, and 7,129,261, and PCT application nos. PCT/US2002/17210, PCT/US2005/017804, PCT/US2006/37793, PCT/US2006/060050, PCT/US2006/060711, WO2006/110476, and U.S. patent application No. 60/891,028, each of which is incorporated herein by reference in its entirety. For further discussion of the type of cytotoxin, linker and methods for coupling a therapeutic agent to an antibody, see also Saito, G. et al (2003) adv. drug Deliv. Rev.55: 199-215; trail, P.A. et al (2003) Cancer Immunol.Immunother.52: 328-337; payne, G. (2003) Cancer Cell 3: 207-; allen, T.M. (2002) nat. Rev. cancer2: 750-; pastan, I, and Kreitman, R.J, (2002) curr, Opin, Investig, drugs3: 1089-; senter, P.D. and Springer, C.J. (2001) adv. drug Deliv. Rev.53: 247-264.

The antibodies may also be conjugated with radioisotopes to produce cytotoxic radiopharmaceuticals, also known as radioimmunoconjugates. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine 131, indium 111, yttrium 90, and lutetium 177. Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available and include

(IDEC Pharmaceuticals) and

(Corixa Pharmaceuticals) and similar methods can be used to prepare radioimmunoconjugates using the antibodies disclosed herein.

The antibody conjugates disclosed herein can be used to modulate a given biological response, and the drug moiety should not be construed as limited to classical chemotherapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin a, pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon-gamma; or a biological response modifier, such as a lymphokine, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factor.

Techniques For coupling the therapeutic moiety to an antibody are well known, see, e.g., Arnon et al, "Monoclonal Antibodies For Immunotargeting drugs In Cancer Therapy" (Monoclonal Antibodies For Immunotargeting drugs In Cancer Therapy), described In Monoclonal Antibodies and Cancer Therapy (Monoclonal Antibodies and Cancer Therapy), Reisfeld et al (ed.), pp. 243-56 (Alan R.Liss Co., Ltd., 1985); hellstrom et al, "Antibodies For Drug Delivery" (Controlled Drug Delivery) (2 nd edition), Robinson et al (ed.), pages 623-53 (Marcel Dekker, Inc. 1987); thorpe, "(Antibody vector Review For Cytotoxic Agents In Cancer Therapy: A Review)," described In Monoclonal Antibodies '84: Biological And clinical Applications (Monoclonal Antibodies'84: Biological And clinical Applications, Pincher et al (ed.), page 475 @ 506 (1985), "Analysis, Results And Future expansion Of Therapeutic Use Of Radiolabeled Antibodies In Cancer Therapy (Analysis, Results, And Future) (Monoclonal Antibodies For Cancer Detection And Therapy) (Monoclonal Antibodies Analysis, repair, And repair, tissue diagnosis, Cancer Therapy, repair, fire Of radial Antibody In Therapy), written In Monoclonal Antibodies For Cancer Detection And Therapy (19816, published by 58-58, et al, 1985).

Bispecific molecules

In another aspect, bispecific molecules comprising anti-BST 1 antibodies or fragments thereof can be used. The antibody or antigen-binding portion thereof can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or a ligand for a receptor) to produce a bispecific molecule that binds to at least two different binding sites or target molecules. Indeed, the antibodies disclosed herein may be derivatized or linked to more than one other functional molecule to produce multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be included in the term "bispecific molecule" as used herein. To produce a bispecific molecule, an antibody can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, in order to produce a bispecific molecule.

Accordingly, the present invention includes bispecific molecules comprising at least one first binding specificity for a first target epitope (i.e., BST1) and a second binding specificity for a second target epitope. The second target epitope can be present on the same target protein as the target protein to which the first binding specificity binds; or the second target epitope may be present on a different target protein than the target protein to which the first binding specificity binds. The second target epitope may be present on the same cell as the first target epitope (i.e., BST 1); or the second target epitope may be present on a target that is not displayed by the cell displaying the first target epitope. The term "binding specificity" as used herein refers to a portion comprising at least one antibody variable domain.

In one embodiment of the invention, the second target epitope is an Fc receptor, such as human fcyri (CD64) or human Fc α receptor (CD 89). accordingly, the present disclosure includes bispecific molecules capable of binding to effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)) expressing fcyr or Fc α R and to target cells expressing BST1 these bispecific molecules target cells expressing BST1 to the effector cells and trigger Fc receptor mediated effector cell activities, such as phagocytosis of cells expressing BST1, antibody dependent cell mediated cytotoxicity (ADCC), cytokine release, or production of superoxide anions.

In another embodiment of the invention, the second target epitope is CD3 or CD 5. Accordingly, the present disclosure includes bispecific molecules capable of binding to effector cells expressing CD3 or CD5 (e.g., cytotoxic T cells expressing CD3 or CD 5) and to target cells expressing BST 1. These bispecific molecules target BST 1-expressing cells to effector cells and trigger CD3 or CD 5-mediated effector cell activities such as T cell clonal expansion and T cell cytotoxicity. In this embodiment, the bispecific antibody can have a total of two or three antibody variable domains, wherein a first portion of the bispecific antibody is capable of recruiting the activity of a human immune effector cell by specifically binding to an effector antigen located on the human immune effector cell, wherein the effector antigen is a human CD3 or CD5 antigen, the first portion consists of one antibody variable domain, and a second portion of the bispecific antibody is capable of specifically binding to a target antigen that is not an effector antigen (e.g., BST1), the target antigen is located on a target cell that is not the human immune effector cell, and the second portion comprises one or two antibody variable domains.

In one embodiment where the bispecific molecule is multispecific, the molecule may further comprise a third binding specificity in addition to the anti-Fc binding specificity or anti-CD 3 or CD5 binding specificity and the anti-BST 1 binding specificity. In one embodiment, the third binding specificity is an anti-Enhancement Factor (EF) moiety, e.g., a molecule that binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell. An "anti-enhancer moiety" can be an antibody, functional antibody fragment or ligand that binds to a given molecule (e.g., an antigen or receptor) and thus results in an enhanced effect of the binding determinant on the Fc receptor or target cell antigen. The "anti-enhancer moiety" can bind to an Fc receptor or a target cell antigen. Alternatively, the anti-enhancer moiety may bind to an entity that is different from the entity to which the first and second binding specificities bind. For example, the anti-enhancer element moiety can bind to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1, or other immune cells that result in an increase in immune response to target cells).

In one embodiment, the bispecific molecule comprises at least one antibody or antibody fragment thereof (including, e.g., Fab ', F (ab')₂Fv, Fd, dAb or single-chain Fv). The antibody may also be a light or heavy chain dimer or any minimal fragment thereof such as an Fv or single chain construct, as described in U.S. Pat. No. 4,946,778, the contents of which are expressly incorporated by reference.

In one embodiment, the binding specificity for the Fc γ receptor is provided by a monoclonal antibody whose binding is not blocked by human immunoglobulin g (igg). As used herein, the term "IgG receptor" refers to any of the 8 γ -chain genes located on chromosome 1. These genes encode a total of 12 transmembrane or soluble receptor isoforms, which fall into three Fc γ receptor classes: fγ RI (CD64), Fc γ RII (CD32) and Fc γ RIII (CD 16). In a preferred embodiment, the Fc γ receptor is a human high affinity Fc γ RI. Human Fc γ RI is a 72kDa molecule that exhibits high affinity for monomeric IgG (10)⁸-10⁹M^-1)。

The generation and characterization of certain preferred anti-Fc γ monoclonal antibodies is described in PCT publication No. WO 88/00052 and U.S. patent No. 4,954,617, the teachings of which are incorporated herein by reference in their entirety. These antibodies bind to an epitope of Fc γ RI, Fc γ RII or Fc γ RIII at a site that is different from the Fc γ binding site of the receptor and, therefore, whose binding is not substantially blocked by physiological levels of IgG. Specific anti-Fc γ RI antibodies useful in the present invention are mAb 22, mAb 32, mAb 44, mAb 62, and mAb 197. The hybridoma producing mAb 32 is available from the american type culture collection under ATCC accession number HB 9469. In other embodiments, the anti-Fc γ receptor antibody is a humanized form of monoclonal antibody 22 (H22). The generation and characterization of the H22 antibody is described in Graziano, R.F. et al (1995) J.Immunol 155(10) 4996-5002 and PCT publication No. WO 94/10332. The cell line producing the H22 antibody was deposited with the american type culture collection under the designation HA022CL1 and HAs the accession number CRL 11177.

In other preferred embodiments, the binding specificity for an Fc receptor is determined by binding to a human IgA receptor (e.g., Fc- α receptor [ Fc α RI (CD89)]) The term "IgA receptor" is intended to include the gene product of an α -gene (Fc α RI) located on chromosome 19, this gene is known to encode several alternatively spliced transmembrane isoforms of 55 to 110kDa, Fc α RI (CD89) is expressed on monocytes/macrophages, eosinophils and neutrophils in a constitutive manner, but not on non-effector cell populationsFc α RI has moderate affinity for IgA1 and IgA2 (about 5X 10)⁷M^-1) The affinity increases upon exposure to cytokines such as G-CSF or GM-CSF [ Morton, H.C. et al (1996) Critical Reviews in Immunology 16: 423-440-]4 Fc α RI-specific monoclonal antibodies have been described, identified as A3, A59, A62 and A77, which bind Fc α RI outside the IgA ligand binding domain [ Monteiro, R.C. et al (1992) J.Immunol.148:1764]。

Fc α RI and Fc RI are preferred trigger receptors for use in bispecific molecules of the invention because they (1) are expressed predominantly on immune effector cells (e.g., monocytes, PMNs, macrophages, and dendritic cells), (2) are expressed at high levels (e.g., 5,000-100,000 per cell), (3) are mediators of cytotoxic activity (e.g., ADCC, phagocytosis), and (4) mediate enhanced antigen presentation of the antigens targeted thereto, including autoantigens.

Antibodies that can be used in bispecific molecules are murine, human, chimeric and humanized monoclonal antibodies.

Bispecific molecules of the present disclosure can be prepared by coupling component binding specificities, such as anti-FcR, anti-CD 3, anti-CD 5, and anti-BST 1 binding specificities, using methods known in the art. For example, the binding specificity of each bispecific molecule can be generated separately and then coupled to each other. When the binding specificity is a protein or peptide, various coupling or crosslinking agents can be used for covalent coupling. Examples of crosslinking agents include protein a, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5' -dithiobis (2-nitrobenzoic acid) (DTNB), o-phenylenebismaleimide (oppdm), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), and sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) [ see, e.g., Karpovsky et al (1984) j.

1686 of 160: 1686; liu, MA et al (1985) Proc.Natl.Acad.Sci.USA 82:8648 ]. Other methods include those described in Paulus (1985) Behring Ins.Mitt. 78, 118-; brennan et al (1985) Science229: 81-83; and Glennie et al (1987) J.Immunol.139: 2367-. Preferred coupling agents are SATA and sulfo-SMCC, both available from Pierce corporation (Rockford, Ill.).

Additional bivalent structures have been obtained for bispecific by Engineering double binding into full-length antibody-like forms (Wu et al, 2007, Nature Biotechnology 25[11]: 1290-1297; USSN12/477,711; Michaelson et al, 2009, mAbs 1[2]: 128-141; PCT/US 2008/074693; Zuo et al, 2000, Protein Engineering 13[5]: 361-367; USSN09/865,198; Shen et al, 2006, J Biol Chem 281[16]: 10706-10714; Lu et al, 2005, J Biol Chem 280[20]: 19665-19672; PCT/US 2005/025472; which are expressly incorporated herein by reference).

When the binding specificity is an antibody, it may be conjugated by thiol bonding of the C-terminal hinge region of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of thiol residues, preferably 1 thiol residue, prior to coupling.

Alternatively, both binding specificities may be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific molecule is mAb x mAb, mAb x Fab, Fab x F (ab')₂Or a ligand x Fab fusion protein. The bispecific molecule may be a single chain molecule comprising one single chain antibody and a binding determinant or a single chain bispecific molecule comprising two binding determinants. A bispecific molecule can comprise at least two single chain molecules. Methods for making bispecific molecules are described, for example, in U.S. Pat. nos. 5,260,203, 5,455,030, 4,881,175, 5,132,405, 5,091,513, 5,476,786, 5,013,653, 5,258,498 and 5,482,858, each of which is expressly incorporated herein by reference.

Binding of a bispecific molecule to its specific target can be demonstrated by, for example, enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), FACS analysis, biological assays (e.g., growth inhibition) or Western blot assays. Typically, each of these assays detects the presence of a particular protein-antibody complex of interest by using a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, the FcR-antibody complex can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antibody-FcR complex. Alternatively, complexes may be detected using any of a variety of other immunoassays. For example, antibodies can be radiolabeled and used in Radioimmunoassays (RIA) (see, e.g., Weintraub, B., radioimmunoassay Principles (Principles), sensing Training Course on radio ligand and Assay Techniques, The endocrinococcity, 3.1986, which is incorporated herein by reference). The radioisotope may be detected by means such as the use of a gamma counter or scintillation counter or by autoradiography.

Antibody fragments and antibody mimetics

The pharmaceutical combination of the present invention is not limited to conventional antibodies and can be practiced through the use of antibody fragments and antibody mimetics. As described in detail below, a variety of antibody fragments and antibody simulation techniques have been developed and are not well known in the art. Although many of these techniques, such as domain antibodies, nanobodies, and unibodies utilize fragments or other modifications of traditional antibody structures, there are still alternative techniques, such as affibodies, darpins, anti-carrier proteins, Avimer, and Versabody that employ binding structures that, while mimicking traditional antibody binding, are generated and act via different mechanisms.

Domain antibodies (dAbs) are the smallest functional binding units of antibodies, corresponding to human antibody heavy chains (V)_H) Or light chain (V)_L) The variable region of (a). The domain antibody has a molecular weight of about 13 kDa. Domantis has developed a large series of fully human V's that are large and highly functional_HAnd V_LdAb libraries (more than 100 million different sequences in each library) and these libraries are used to select dabs specific for therapeutic targets. In contrast to many conventional antibodies, domain antibodies are well expressed in bacterial, yeast and mammalian cell systems. Additional details of domain antibodies and methods of their production can be found in the above-described examples by reference to U.S. patent nos. 6,291,158, 6,582,915, 6,593,081, 6,172,197, 6,696,245; united states is serial No. 2004/0110941; european patent application No. 1433846 and european patents 0368684 and 0616640; WO05/035572, WO04/101790. WO04/081026, WO04/058821, WO04/003019 and WO03/002609, each of which is incorporated by reference in its entirety.

Nanobodies are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy chain antibodies. These heavy chain antibodies contain a single variable domain (VHH) and two constant domains (C)_H2 and C_H3). Importantly, the cloned and isolated VHH domain is an extremely stable polypeptide with the full antigen-binding capacity of the original heavy chain antibody. V of nano antibody and human antibody_HThe domains have high homology and can be further humanized without any loss of activity. Importantly, nanobodies have low immunogenic potential, which has been demonstrated in primate studies using nanobody lead compounds.

Nanobodies combine the advantages of conventional antibodies with the important features of small molecule drugs. Like conventional antibodies, nanobodies exhibit high target specificity for their target, high affinity, and low inherent toxicity. However, like small molecule drugs, they inhibit enzymes and readily access the receptor cleft. In addition, nanobodies are extremely stable, can be administered by means other than injection (see, e.g., WO 04/041867, incorporated herein by reference in its entirety) and are easy to prepare. Other advantages of nanobodies include recognition of unusual or hidden epitopes due to their small size, binding with high affinity and selectivity into the lumen or active site of a protein target due to their unique 3-dimensional structure, drug format flexibility, tunability of half-life, and ease and speed of drug development.

Nanobodies are encoded by a single gene and are produced efficiently in almost all prokaryotic and eukaryotic hosts, such as escherichia coli (see, e.g., US 6,765,087, which is incorporated herein by reference in its entirety), molds (e.g., Aspergillus (Aspergillus) or Trichoderma (Trichoderma)), and yeasts (e.g., saccharomyces, Kluyveromyces (Kluyveromyces), Hansenula (Hansenula), or pichia) (see, e.g., US 6,838,254, which is incorporated herein by reference in its entirety). The production process is scalable and has produced nanobodies in multi-kilogram quantities. Nanobodies may be formulated as long shelf-life ready-to-use solutions because they exhibit superior stability compared to conventional antibodies.

The nano-cloning method (see, e.g., WO 06/079372, which is incorporated herein by reference in its entirety) is a proprietary method of automated high-throughput B-cell-based selection, generating nanobodies against the target, and may be used in the context of the present invention.

Unibody is another antibody fragment technology; however this technique is based on the removal of the hinge region of the IgG4 antibody. The deletion of the hinge region results in a molecule that is substantially half the size of a conventional IgG4 antibody and has a monovalent binding region rather than the bivalent binding region of the IgG4 antibody. It is also well known that IgG4 antibodies are inert and therefore do not interact with the immune system, which can be advantageous for treating diseases where an immune response is undesirable, and this advantage is translated to unibodies. For example, the unibodies may act to inhibit or silence, rather than kill, the cells to which they bind. In addition, the unibodies bound to cancer cells did not stimulate their proliferation. In addition, since the size of the Unibody is about half that of the traditional IgG4 antibody, it can show better distribution over larger solid tumors and produce potentially beneficial efficacy. Unibodies clear from the body at a rate similar to that of the intact IgG4 antibody and are able to bind their antigen with an affinity similar to that of whole antibodies. Additional details of the Unibody can be obtained by reference to patent publication WO2007/059782, which is incorporated herein by reference in its entirety.

The three-helix bundle domain has been used as a backbone for constructing a combinatorial phage library from which Affibody variants targeting the desired molecule [ Nord K, Guinneusson E, Ringdahl J, Stahl S, Uhlenn M, Nygren PA, (1997) "selected from Binding proteins of the α -helix bacterial receptor domain combinatorial library (Binding Protein selected Binding partners of the library of America Biotechnology of α -helicobacter receptor), Nature Biotech 15: 772-7; Ronddymark J, GronluH, Uygen PA (2002) derived from the Protein A combinatorial engineered Human Protein A, the expression of Binding Protein, IgG, Binding Protein, IgG, CD-Binding Protein, IgG, and other antibody, IgG, Binding Protein.

The labeled affibodies can also be used in imaging applications for determining the abundance of isoforms.

DARPin (designed ankyrin repeat protein) is an example of an antibody mimetic DRP (designed repeat protein) technology that has been developed to explore the ability of non-antibody polypeptides to bind. Repeat proteins such as ankyrin or leucine rich repeat proteins are common binding molecules, which, unlike antibodies, exist both intracellularly and extracellularly. Its unique modular architecture features repeating structural units (repeats) that are stacked together to form an extended repeat domain that exhibits a variable and modular target binding surface. Based on this modularity, combinatorial libraries of polypeptides with highly diverse binding specificities can be generated. This strategy involves the consensus design of self-compatible repeats that display variable surface residues and their random assembly into repeat domains.

Darpins can be produced in very high yields in bacterial expression systems and belong to the most stable proteins known. Darpins have been selected for high specificity and high affinity against a wide range of target proteins including human receptors, cytokines, kinases, human proteases, viruses and membrane proteins. Darpins with affinities ranging from a few nanomolar to picomolar units can be obtained.

Darpins have been used in a wide variety of applications including ELISA, sandwich ELISA, flow cytometry analysis (FACS), Immunohistochemistry (IHC), chip applications, affinity purification, or Western blotting. Darpins have also been shown to be highly active in intracellular compartments, for example as intracellular marker proteins fused to Green Fluorescent Protein (GFP). DARPin was further used to inhibit viral entry with a pM range of IC 50. Darpins not only ideally block protein-protein interactions, but also inhibit enzymes. Proteases, kinases and transporters have been successfully inhibited, most often in an allosteric inhibition mode. The extremely rapid and specific enrichment on tumors and the extremely favorable tumor-to-blood ratio make darpins particularly suitable for in vivo diagnostics or therapeutic methods.

Additional information regarding darpins and other DRP techniques can be found in U.S. patent application publication No. 2004/0132028 and international patent publication No. WO 02/20565, each of which is incorporated by reference in its entirety.

However, in this case, the binding specificity derives from lipocalin (lipocalin), a family of low molecular weight proteins that are naturally and abundantly expressed in human tissues and body fluids.lipocalin has evolved to perform a range of functions in vivo related to physiological transport and storage of chemically sensitive or insoluble compounds.lipocalin has a robust, intrinsic structure comprising a highly conserved β -barrel supporting 4 loops at one end of the protein.these loops form the entrance of the binding pocket, and conformational differences in this part of the molecule account for variations in binding specificity between individual lipocalins.

Although the overall structure of the conserved β -fold framework-supported hypervariable loops is similar to an immunoglobulin, the lipocalins differ significantly in size from antibodies and consist of a single polypeptide chain of 160-180 amino acids, which is slightly larger than a single immunoglobulin domain.

The lipocalin is cloned and its loop is engineered to produce the anti-transporter. Libraries of structurally diverse anti-cargo proteins have been generated, and anti-cargo protein display allows selection and screening for binding function, followed by expression and generation of soluble proteins for further analysis in prokaryotic or eukaryotic systems. Studies have successfully demonstrated that an antiporter protein can be formed that is specific for almost any human target protein, that can be isolated, and that binding affinities in the nanomolar or higher range can be obtained.

The antiporter protein may also be configured as a dual targeting protein, so-called Duocalin. Duocalin binds two separate therapeutic targets in one monomeric protein that is readily produced using standard manufacturing processes, while retaining target specificity and affinity, regardless of the structural orientation of its two binding domains.

Modulation of multiple targets via a single molecule is particularly advantageous in diseases known to involve more than a single etiologic agent. In addition, bivalent or multivalent binding forms such as Duocalin have significant potential in: targeting cell surface molecules in the disease, mediating agonism on signal transduction pathways, or inducing enhanced internalization via binding and clustering of cell surface receptors. In addition, the high intrinsic stability of Duocalin is comparable to monomeric anti-carrier proteins, providing flexible formulation and delivery potential for Duocalin.

Additional information regarding anti-transporters can be found in U.S. Pat. No. 7,250,297 and International patent publication No. WO99/16873, each of which is incorporated by reference in its entirety.

Another antibody mimetic technology that can be used in the context of the present invention is Avimer. Avimer evolved from a large family of human extracellular receptor domains by exon shuffling and phage display in vitro, resulting in multi-domain proteins with binding and inhibitory properties. It has been shown that the attachment of multiple independent binding domains results in avidity and improved affinity and specificity compared to conventional single epitope binding proteins. Other potential advantages include simple and efficient production of multi-target specific molecules in E.coli, improved thermostability and resistance to proteases. Avimer with affinity lower than nanonemolar to a variety of targets has been obtained.

Additional information regarding Avimer may be found in U.S. patent application publication nos. 2006/0286603, 2006/0234299, 2006/0223114, 2006/0177831, 2006/0008844, 2005/0221384, 2005/0164301, 2005/0089932, 2005/0053973, 2005/0048512, 2004/0175756, each of which is incorporated by reference in its entirety.

Versabody is another antibody mimetic technology that can be used in the context of the present invention. Versabody is a small 3-5kDa protein with > 15% cysteine, which forms a high disulfide bond density backbone replacing the hydrophobic core typical of proteins. Replacement of a large number of hydrophobic amino acids (comprising a hydrophobic core) with a few disulfide bonds results in proteins that are smaller, more hydrophilic (less aggregation and non-specific binding), more resistant to proteases and heat, and have a lower T cell epitope density, since the residues that make the greatest contribution to MHC presentation are hydrophobic. It is well known that all 4 of these properties affect immunogenicity and are expected to together cause a dramatic reduction in immunogenicity.

Versabody's inspiration comes from natural injectable biopharmaceuticals produced by leeches, snakes, spiders, scorpions, snails and sea anemones, which are known to exhibit unexpectedly low immunogenicity. Starting from a selected family of native proteins, size, hydrophobicity, proteolytic antigen processing and epitope density are minimized by design and screening to a degree well below the mean of native injectable proteins.

Given the structure of Versabody, these antibody mimetics provide a common paradigm that includes multivalent, multispecific, diverse half-life mechanisms, tissue targeting modules, and the absence of an antibody Fc region. In addition, Versabody is produced in high yield in escherichia coli, and because of its hydrophilicity and small size, Versabody is highly soluble and can be formulated in high concentrations. Versabody has unexpected thermal stability (it can be boiled) and provides extended shelf life.

Additional information regarding Versabody can be found in U.S. patent application publication No. 2007/0191272, which is incorporated by reference in its entirety.

The detailed descriptions of antibody fragments and antibody mimetic techniques provided above are not intended to be a comprehensive list of all techniques that can be used in the context of this specification. For example, and without limitation, a variety of additional techniques may be used in the context of the present invention, including polypeptide-based alternative techniques, such as the fusion of complementarity determining regions outlined in, for example, Qui et al (2007) Nature Biotechnology 25(8):921-929, which is incorporated by reference in its entirety; and nucleic acid-based techniques such as the RNA aptamer techniques described in U.S. patent nos. 5,789,157, 5,864,026, 5,712,375, 5,763,566, 6,013,443, 6,376,474, 6,613,526, 6,114,120, 6,261,774, and 6,387,620, each of which is incorporated by reference in its entirety.

Pharmaceutical composition

The pharmaceutical combinations of the present invention are in the form of a combined preparation for simultaneous, separate or sequential use. Similarly, in the methods of the invention, components (a) and (B) of the pharmaceutical combination may be administered to the patient simultaneously, separately or sequentially.

The term "combined preparation" includes both fixed and non-fixed combinations. The term "fixed combination" means that the active ingredients, e.g. components (a) and (B), are in a single entity or dosage form. In other words, the active ingredients are present in a single composition or formulation. The term "non-fixed combination" means that the active ingredients, e.g. components (a) and (B), are present in different entities or doses (e.g. in separate compositions or formulations), e.g. in the form of kits of parts. The separate components (a) and (B) (in their desired compositions or formulations) may then be administered separately or sequentially at the same time point or at different time points.

Where administration is sequential, the delay in administering the second component should not result in a benefit that would detract from the effect produced by the use of the combination. Thus, in one embodiment, sequential treatment involves administration of the components of the combination over a period of 11 days. In another embodiment, this period of time is 10 days. In another embodiment, this period of time is 9 days. In another embodiment, this period of time is 8 days. In another embodiment, this period of time is 7 days. In another embodiment, this period of time is within 6 days. In another embodiment, this period of time is within 5 days. In another embodiment, this period of time is within 4 days. In another embodiment, this period of time is within 3 days. In another embodiment, this period of time is within 2 days. In another embodiment, this period of time is within 24 hours. In another embodiment, this period of time is within 12 hours.

Components (a) and (B) may be administered in any order, e.g., component (a) is administered first and component (B) is administered subsequently; or component (B) is administered first and component (A) is administered subsequently.

The ratio of the total amounts of component (a) to component (B) to be administered in the combined preparation may be varied, for example, in order to cope with the needs of a patient sub-population to be treated or the needs of individual patients, said different needs being due to age, sex, body weight, etc. of the patients.

Components (a) and (B) present in a single composition or separate compositions may be formulated independently with one or more pharmaceutically acceptable carriers. The pharmaceutical combination of the invention may also comprise at least one other anti-tumour or anti-inflammatory agent or immunosuppressant agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on the use of the antibodies disclosed herein.

Such a combination may comprise one antibody or bispecific molecule disclosed herein or a combination of (e.g., two or more different) antibodies or bispecific molecules disclosed herein. For example, a pharmaceutical combination of the invention may comprise a combination of antibodies (or bispecific molecules) that bind to different epitopes on the target antigen or have complementary activity.

The pharmaceutical combination of the invention may also be administered in combination therapy, i.e. in combination with other agents. For example, a combination therapy may comprise an antibody of the invention in combination with at least one other anti-neoplastic or anti-inflammatory agent or immunosuppressive agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on the use of the antibodies of the invention.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody, immunoconjugate or bispecific molecule, may be encapsulated in a material that protects the compound from the action of acids and other natural conditions that might inactivate the compound.

Components (A) and (B) of the present invention may comprise one or more pharmaceutically acceptable salts. "pharmaceutically acceptable salt" refers to a salt that retains the biological activity of the desired parent compound and does not cause any undesirable toxicological effects [ see, e.g., Berge, s.m. et al (1977) j.pharm.sci.66:1-19 ]. Examples of the salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like; and those derived from non-toxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like; and those derived from nontoxic organic amines such as N, N' -dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like, (2) oil-soluble antioxidants such as ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), lecithin, propyl gallate, α -tocopherol, and the like, and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may be used in the pharmaceutical compositions of the invention (components (a) and/or (B)) include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Proper fluidity can be maintained, for example, by the use of a coating material, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These combinations (components (a) and/or (B)) may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of the presence of microorganisms can be ensured by the sterilization process as before and by the inclusion of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, and the like). It may also be desirable to incorporate isotonic agents (such as sugars, sodium chloride, and the like) into the composition. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use of the pharmaceutical combination of the invention is contemplated. Supplementary active compounds may also be incorporated into the compositions.

Therapeutic combinations must generally be sterile and stable under the conditions of manufacture and storage. The combination (components (a) and/or (B)) may be formulated as a solution, microemulsion, liposome or other ordered structure suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. In many cases, it will be preferred to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount of the composition which produces a therapeutic effect. Generally, this amount may be from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%, by 100% in combination with a pharmaceutically acceptable carrier.

The dosing regimen (of components (a) and/or (B)) is adjusted to provide the desired optimal response (e.g. synergistic combination, therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the doses may be proportionally reduced or increased as indicated by the need for a treatment condition. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the unit dosage form of the invention is determined and directly dependent on: (a) the unique characteristics of the active compounds and the particular therapeutic effect to be achieved, and (b) the inherent limitations within the art of synthesizing such active compounds for the treatment of sensitivity in an individual.

Preferably, the combination of components (a) and (B) is a synergistic combination. It will be understood by those skilled in the art that a synergistic combination is one in which the effect of the combination is greater than the sum of the effects of the individual components. Synergy can be quantified using the Chou-Talalay Combinatorial Index (CI) (see "assessment of combination chemotherapy: integration of nonlinear regression, curve-shift, isobologram, and combinatorial index analysis (Evaluation of combination chemotherapy: integration of integration chemotherapy of nonlinear regression, current shift, isobologram, and combination indexes)", Zhoo L et al client research Res (2004) 12.1.; 10(23): 7994-8004; and "computerized quantification of synergy and antagonism of paclitaxel, topotecan, and cisplatin on human teratoma cell growth: rational method of clinical protocol design (synergy of aggregation and aggregation of taxol, topotecan, and clinical diagnosis of teratocarcinoma cell growth: 20. J. (9.20.) see" assessment of combination chemotherapy, equilibrium, clinical analysis, 11. J. (9.) (9. J.), (9. 20). The Combination Index (CI) method is based on a multi-drug effect equation derived from the principle of median effect in the law of mass action. This provides a quantitative definition of strong synergy (CI <0.3), synergy (CI ═ 0.3-0.9), additive effect (CI ═ 0.9-1.1), or antagonism/no benefit (CI >1.1), and it provides algorithms to computer software for automated simulation of drug combinations. Which takes into account the potency (d (m) value) and the shape (m value) of the dose-effect curve of each drug separately and in combination. The Chou-Talalay Combination Index (CI) can be estimated using the Synergy R kit (see "Preclinical drug Combination Studies", Chou TC. Leuk. Lymphoma. (2008); 49(11): 2059-. The CI combined may be tested in a suitable cell line, for example in K052 cells, for example under the conditions used in example 9.

Preferably, the pharmaceutical combination of the invention is a synergistic combination having a Chou-Talalay Combination Index (CI) of less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2. Preferably, CI is 0.1-0.5, 0.1-0.3, or 0.1-0.2.

In particular, a method of treating cancer in a patient is provided, comprising administering simultaneously, sequentially or separately to a patient in need thereof a therapeutically effective synergistic amount of components (a) and (B) of the pharmaceutical combination of the invention. Also provided is a pharmaceutical combination of the invention for use in the treatment of cancer, wherein components (a) and (B) are administered to a patient simultaneously, separately or sequentially in synergistic amounts in order to treat cancer. Preferably, the amounts of components (a) and (B) are administered to the patient in order to provide the plasma concentrations disclosed above.

Also provided is the use of a synergistic amount of components (a) and (B) of the pharmaceutical combination of the invention for the preparation of a pharmaceutical combination for simultaneous, separate or sequential use in the treatment of cancer. Also provided is a synergistic pharmaceutical combination of the invention for use in therapy or as a medicament.

For administration of the antibody, the dosage range is about 0.0001 to 100mg/kg, and more typically 0.01 to 5mg/kg of host body weight. For example, the dose may be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, or 10mg/kg body weight or in the range of 1-10mg/kg body weight. Exemplary treatment regimens entail administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3 to 6 months. A preferred dosing regimen for the anti-BST 1 antibodies of the invention includes 1mg/kg body weight or 3mg/kg body weight via intravenous administration and the antibodies are administered using one of the following dosing regimens: (i) six doses were continued every four weeks, followed by every three months; (ii) every three weeks; (iii) once at 3mg/kg body weight, then 1mg/kg body weight every three weeks.

In some embodiments, the anti-BST 1 antibody (e.g., BST1_ a2) dose is adjusted to achieve a plasma antibody concentration of 0.005 to 50 μ g/ml, e.g., 0.01 to 10 μ g/ml. Preferably, the anti-BST 1 antibody (e.g., BST1_ a2) dose is adjusted to achieve a plasma antibody concentration of 0.01 to 0.1 μ g/ml, 0.1 to 1.0 μ g/ml, or 1.0 to 10 μ g/ml.

In some embodiments, the cytidine analog (e.g., 5-azacytidine) dose is adjusted to achieve a plasma concentration of 0.05 to 5 μ Μ, e.g., 0.1 to 2 μ Μ. Preferably, the cytidine analog dose is adjusted to achieve a plasma concentration of 0.1 to 0.5 μ M, 0.5 to 1.0 μ M, or 1.0 to 2.0 μ M.

In some embodiments, the cytidine analog (e.g., decitabine) dose is adjusted to achieve a plasma concentration of 0.05 to 5 μ Μ, e.g., 0.1 to 2 μ Μ. Preferably, the cytidine analog dose is adjusted to achieve a plasma concentration of 0.1 to 0.5 μ M, 0.5 to 1.0 μ M, or 1.0 to 2.0 μ M.

A cytidine analog (e.g., 5-azacytidine or decitabine), or a pharmaceutically acceptable salt thereof, may be administered with one or more of the following: cyclophosphamide, hydroxydaunomycin, ancepin, and prednisone or prednisolone (i.e., CHOP therapy).

In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered falls within the indicated range. The antibody is typically administered multiple times. The time interval between individual doses may be, for example, weekly, monthly, every three months, or yearly. The time intervals may also be irregular, as indicated by measuring blood levels of antibodies to the target antigen in the patient. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1-1000 μ g/ml, and in some methods about 25-300 μ g/ml.

Alternatively, the components may be administered in a sustained release formulation, in which case less frequent administration is required. The dose and frequency will vary depending on the half-life of the antibody in the patient. Typically, human antibodies exhibit the longest half-life, followed by humanized, chimeric, and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at relatively infrequent intervals over a long time frame. Some patients continue to receive treatment for the remainder of the life. In therapeutic applications, relatively high doses at relatively short time intervals are sometimes required until progression of the disease is reduced or terminated, and preferably until the patient exhibits partial or complete amelioration of the symptoms of the disease. Thereafter, a prophylactic regimen may be administered to the patient.

The actual dosage level of the active ingredients in the pharmaceutical combination of the present invention can be varied so as to obtain an amount of the active ingredients which is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, and which is non-toxic to the patient. The selected dosage depends upon a variety of pharmacokinetic factors including the activity of the particular combination of the invention or ester, salt or amide thereof employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or materials used in conjunction with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

Preferably, a "therapeutically effective dose" of an anti-BST 1 antibody results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of damage or disability due to disease invasion. For example, to treat a BST 1-mediated tumor, a "therapeutically effective dose" preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80%, relative to an untreated subject. The ability of a compound to inhibit tumor growth can be assessed in an animal model system that predicts efficacy in human tumors. Alternatively, this property of the composition can be assessed by testing the ability of the compound to inhibit cell growth, such inhibition being measured in vitro by assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise improve the symptoms of a subject. One of ordinary skill in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

Components (A) and (B) of the present invention may be administered via one or more routes of administration using one or more of a variety of methods known in the art; the routes can be the same or different for components (A) and (B). As will be appreciated by those skilled in the art, the route and/or mode of administration will vary depending on the desired result. Preferred routes of administration for the antibody include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, e.g., by injection or infusion. Preferably, the pharmaceutical combination is administered intravenously.

As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subdermal, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

Alternatively, components (a) and (B) may be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g. intranasal, oral, vaginal, rectal, sublingual or topical.

Components (a) and (B) may be prepared together with carriers that protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Various methods for preparing such formulations have been patented or are generally known to those skilled in the art [ see, e.g., Sustained and Controlled Release Drug Delivery Systems (1978) J.R. Robinson, Marcel Dekker GmbH, N.Y. ].

The drug combinations may be administered together or separately using medical devices known in the art. For example, in a preferred embodiment, the drug combinations of the present invention can be administered using a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824 or 4,596,556. Examples of well-known implants and modules that may be used in the present invention include: U.S. patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing a drug at a controlled rate; U.S. patent No. 4,486,194, which discloses a therapeutic device for transdermal administration of a drug; U.S. Pat. No. 4,447,233, which discloses a drug infusion pump for delivering a drug at a precise infusion rate; U.S. patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous delivery of a drug; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multiple lumen compartments; and U.S. patent No. 4,475,196, which discloses osmotic drug delivery systems. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the monoclonal antibodies disclosed herein can be formulated to ensure proper distribution in vivo. For example, the Blood Brain Barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compound crosses the BBB (if desired), it can be formulated, for example, in liposomes. For methods of preparing liposomes, see, e.g., U.S. Pat. nos. 4,522,811, 5,374,548, and 5,399,331. Liposomes can comprise one or more moieties that are selectively transported into a particular cell or organ, thereby enhancing targeted drug delivery [ see, e.g., v.v. ranade (1989) j.clin.pharmacol.29:685 ]. Exemplary targeting moieties include folic acid or biotin (see, e.g., U.S. Pat. No. 5,416,016); mannoside [ Umezawa et al (1988) biochem. Biophys. Res. Commun.153:1038 ]; antibodies [ P.G.Bloeman et al (1995) FEBSLett.357: 140; M.Owais et al (1995) Antimicrob.Agents Chemother.39: 180; the surfactant protein A receptor [ Briscoe et al (1995) am.J.Physiol.1233:134 ]; p120[ Schreier et al (1994) J.biol.chem.269:9090 ]; see also k.keinanen; M.L.Laukkanen (1994) FEBS Lett.346: 123; j.j.killion; fidler (1994) Immunomethods 4: 273.

Use and method

The pharmaceutical combinations and methods disclosed herein have a number of in vivo therapeutic uses, including the treatment of BST 1-mediated diseases.

In some embodiments, these combinations can be administered (e.g., in vivo) to a subject to treat a variety of disorders. As used herein, the term "subject" is intended to include both human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. Preferred subjects include human patients suffering from conditions mediated by BST1 activity. The methods are particularly useful for treating human patients having a disorder associated with aberrant BST1 expression. When the antibody to BST1 is administered with another agent, the two can be administered in any order or simultaneously.

In addition, in view of the expression of BST1 on tumor cells, the pharmaceutical combination of the present invention can be used to treat subjects suffering from tumorigenic diseases, such as diseases characterized by the presence of tumor cells expressing BST1, including, for example, Acute Myeloid Leukemia (AML), B-cell chronic lymphocytic leukemia, breast cancer, colorectal cancer, kidney cancer, head and neck cancer, lung cancer, ovarian cancer, and pancreatic cancer. BST1 has been shown to be internalized upon antibody binding, as shown in example 5 below, thus making the antibodies of the invention useful in any payload mechanism, e.g., ADC methods, radioimmunoconjugates, or ADEPT methods.

In one embodiment, the combination may be used to inhibit or block BST1 function, which in turn may be associated with the prevention or amelioration of symptoms of certain diseases, thereby suggesting BST1 as a mediator of the disease. This can be achieved by contacting the sample and control sample with an anti-BST 1 antibody under conditions that allow for the formation of a complex between the antibody and BST 1. Any complexes formed between the antibodies and BST1 were detected and compared in the samples and controls.

In another embodiment, the combinations comprising antibodies (e.g., monoclonal antibodies, multispecific and bispecific molecules and compositions) disclosed herein can be preliminarily tested for binding activity associated with in vitro therapeutic uses. For example, the pharmaceutical combinations of the present invention can be tested using the flow cytometry assays described in the examples below.

The combinations comprising antibodies (e.g., monoclonal antibodies, multispecific and bispecific molecules, immunoconjugates and compositions) disclosed herein have additional uses in the treatment of BST 1-related diseases. For example, a combination comprising a monoclonal antibody, a multispecific or bispecific molecule, and an immunoconjugate may be used to elicit in vivo or in vitro one or more of the following biological activities: inhibiting growth and/or killing of cells expressing BST 1; mediate phagocytosis or ADCC of cells expressing BST1 in the presence of human effector cells, or block binding of BST1 ligand to BST 1.

In a particular embodiment, combinations comprising antibodies (e.g., monoclonal antibodies, multispecific and bispecific molecules and compositions) are used in vivo to treat or prevent a variety of BST 1-related diseases. Examples of BST 1-related diseases include, inter alia, human cancer tissue representing Acute Myeloid Leukemia (AML), B-cell chronic lymphocytic leukemia, breast cancer, colorectal cancer, kidney cancer, head and neck cancer, lung cancer, ovarian cancer and pancreatic cancer.

Suitable routes for in vivo administration of components (a) and (B) (e.g., monoclonal antibodies, multispecific and bispecific molecules or immunoconjugates) of the pharmaceutical combinations of the invention are well known in the art and can be selected by one of ordinary skill in the art. For example, the pharmaceutical combination may be administered by injection (e.g., intravenously or subcutaneously). The appropriate dosage of the molecule used will depend on the age and weight of the subject and the concentration and/or formulation of the antibody and cytidine analog compositions.

The pharmaceutical combination of the invention may be co-administered with one or more other therapeutic agents, such as a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent. The antibody may be linked to the agent (as an immune complex) or may be administered separately from the agent. In the latter case (separate administration), the antibody may be administered before, after or simultaneously with the agent or may be co-administered with other known therapies (e.g., anti-cancer therapies, such as radiation). The therapeutic agents include, inter alia, antineoplastic agents that are effective themselves only at levels that are toxic or sub-toxic to the patient, such as doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil, and cyclophosphamide hydroxyurea. Cisplatin was administered intravenously 1 time every 4 weeks at a dose of 100mg/kg, and adriamycin was administered intravenously 1 time every 21 days at a dose of 60-75 mg/ml.

Other agents suitable for co-administration in combination with the agents of the invention include other agents for the treatment of, for example, Acute Myeloid Leukemia (AML), B-cell chronic lymphocytic leukemia, breast cancer, colorectal cancer, renal cancer, head and neck cancer, lung cancer, ovarian cancer or pancreatic cancer, such as

5FU and gemcitabine.

Co-administration of the anti-BST 1 antibodies or antigen-binding fragments thereof disclosed herein with a chemotherapeutic agent (e.g., a cytidine analog) provides two anti-cancer agents that act via different mechanisms that produce cytotoxic effects on human tumor cells. Such co-administration can solve problems caused by tumor cells developing drug resistance or antigenic changes thereof, which can result in tumor cells not responding to antibodies.

Target-specific effector cells, such as effector cells linked to a combination disclosed herein (e.g., comprising monoclonal antibodies, multispecific and bispecific molecules), can also be used as therapeutic agents. The effector cells for targeting may be human leukocytes such as macrophages, neutrophils or monocytes. Other cells include eosinophils, natural killer cells and other cells that carry IgG or IgA receptors. If desired, the effector cells may be obtained from the subject to be treated. Target-specific effector cells can be administered as a suspension of cells in a physiologically acceptable solution. The number of cells administered may be about 10⁸-10⁹But will vary depending on the purpose of the treatment. Typically, this amount will be sufficient to obtain localization at the target cell (e.g., a tumor cell expressing BST 1) and to affect cell killing by, for example, phagocytosis. The route of administration may also vary.

Therapy with target-specific effector cells may be performed in conjunction with other techniques for removing targeted cells. For example, anti-tumor therapies using the pharmaceutical combinations of the invention (e.g., comprising monoclonal antibodies, multispecific or bispecific molecules) and/or effector cells armed with these combinations can be used in combination with chemotherapy. In addition, combination immunotherapy can be used to direct two different cytotoxic effector populations to tumor cell rejection. For example, an anti-BST 1 antibody linked to anti-Fc- γ RI or anti-CD 3 can be used in combination with an IgG or IgA receptor specific binding agent.

The bispecific and multispecific molecules disclosed herein may also be used to modulate Fc γ R or Fc γ R levels on effector cells, such as by capping and ablating receptors on the cell surface. Mixtures of anti-Fc receptors may also be used for this purpose.

The drug combinations of the invention (e.g. comprising monoclonal antibodies, multispecific or bispecific molecules and immunoconjugates) having a complement binding site (such as from a portion of IgG1, IgG2, or IgG3, or IgM that binds complement) may also be used in the presence of complement. In one embodiment, ex vivo treatment of a cell population comprising target cells with a binding agent disclosed herein and appropriate effector cells may be supplemented by the addition of complement or complement-containing serum. Phagocytosis of target cells coated with a binding agent of the invention can be improved by binding to complement proteins. In another embodiment, target cells coated with the compositions disclosed herein (e.g., monoclonal antibodies, multispecific and bispecific molecules) can also be lysed by complement. In yet another embodiment, the combination of the invention does not activate complement.

The pharmaceutical combination of the invention (e.g. comprising a monoclonal antibody, a multispecific or bispecific molecule, or an immunoconjugate) may also be administered with complement. In certain embodiments, the invention provides pharmaceutical combinations comprising an antibody, a multispecific or bispecific molecule, and serum or complement. These compositions may be advantageous when the complement is in close proximity to an antibody, multispecific, or bispecific molecule. Alternatively, the antibodies, multispecific or bispecific molecules, and complement or serum disclosed herein can be administered separately.

Also within the scope of the invention are kits comprising a pharmaceutical combination of the invention (e.g., comprising a monoclonal antibody, a bispecific or multispecific molecule, or immunoconjugate) and instructions for use. The kit may further contain one or more other agents, such as an immunosuppressive agent, a cytotoxic agent, or a radiotoxic agent, or one or more other antibodies disclosed herein (e.g., an antibody having complementary activity to bind to an epitope in BST1 antigen that is distinct from the first antibody).

Thus, another therapeutic agent (other than a cytidine analog) that enhances or enhances the therapeutic effect of an antibody, such as a cytotoxic agent or a radiotoxic agent, can be additionally administered (prior to, concurrently with, or after administration of the antibody disclosed herein) to a patient treated with the pharmaceutical combination of the invention.

In other embodiments, the subject may be additionally treated with an agent that modulates (e.g., enhances or inhibits) the expression or activity of Fc γ or Fc γ receptors by, for example, treating the subject with a cytokine. Preferred cytokines to be administered during treatment with multispecific molecules include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), and Tumor Necrosis Factor (TNF).

The pharmaceutical combinations of the invention (e.g. comprising antibodies, multispecific or bispecific molecules) may also be used to target cells expressing Fc γ R or BST1, e.g. for labelling such cells. For this use, the binding agent may be linked to a molecule that can be detected. Accordingly, the present disclosure provides methods for ex vivo or in vitro localization of cells expressing Fc receptors (such as Fc γ R) or BST 1. The detectable label can be, for example, a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.

In other embodiments, the invention provides methods for treating a BST 1-mediated disorder, such as human cancer and human inflammatory diseases, including the diseases of the invention, in a subject.

In yet another embodiment, the inventive pharmaceutical combinations comprising immunoconjugates disclosed herein can be used to target a compound (e.g., a therapeutic agent, a label, a cytotoxin, a radiotoxin, an immunosuppressant, etc.) to a cell having a BST1 cell surface receptor by linking the compound to an antibody. For example, anti-BST 1 antibodies can be conjugated to any of the toxin compounds described in U.S. patent nos. 6,281,354 and 6,548,530, U.S. patent publication nos. 2003/0050331, 2003/0064984, 2003/0073852, and 2004/0087497, or disclosed in WO 03/022806. Accordingly, the present disclosure provides methods of locating cells expressing BST1 ex vivo or in vivo (e.g., using a detectable label, such as a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor). Alternatively, immunoconjugates can be used to kill cells with BST1 cell surface receptors by targeting a cytotoxin or a radiotoxin to BST 1.

All references cited in this specification, including but not limited to all papers, patent publications, patents, patent applications, lectures, texts, reports, manuscripts, brochures, books, internet postings, journal articles, periodicals, product data sheets, and the like, are hereby incorporated by reference in their entirety. The discussion of the references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art, and applicants reserve the right to challenge the accuracy and pertinence of the cited references.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

The invention is further illustrated by the following examples, which should not be construed as further limiting.

Example 1: construction of phage display libraries

A recombinant protein consisting of amino acids 29-292 of BST1 (SEQ ID NO:44) was synthesized by standard recombinant methods in a eukaryotic manner and used as an antigen for immunization.

Immunization and mRNA isolation

Phage display libraries for identifying BST1 binding molecules were constructed as follows. A/J mice (Jackson laboratories, Balport, Maine) were immunized intraperitoneally with recombinant BST1 antigen (extracellular domain) using 100. mu.g of protein in Freund's complete adjuvant on day 0 and 100. mu.g of antigen on day 28. Via puncture of the posterior orbital sinusTest blood of the mice was obtained. If, by testing the titer, use is made of avidin (reaction-Bind) which is neutral^TM) NeutrAvidin (TM) coated polystyrene plates (Pierce Corp., Rockford, Ill.) immobilized biotinylated BST1 antigen, these titers were considered high by ELISA, and mice were boosted with 100 μ g protein on days 70, 71, and 72, and then sacrificed and splenectomy performed on day 77. If the titer of the antibody is deemed unsatisfactory, mice are boosted with 100 μ g of antigen on day 56 and test blood is taken on day 63. If satisfactory titers were obtained, animals were boosted with 100 μ g of antigen on days 98, 99 and 100 and spleens were harvested on day 105.

Spleens were harvested in a laminar flow hood and transferred to a petri dish, and the fat and connective tissue were sheared and discarded. In the presence of 1.0ml of solution D (25.0g guanidine thiocyanate (Boehringer Mannheim, Indianapolis, Ind.), 29.3ml sterile water, 1.76ml 0.75M sodium citrate pH 7.0, 2.64ml 10% sarcosyl (sarkosyl) (Fisher Scientific, Pittsburgh, Pa., Tex., Philippi., Pennsylvania) was rapidly mashed with a plunger from a sterile 5 syringe, the spleen suspension was aspirated with a 18-gauge needle until all cells were lysed, and the viscous solution was transferred to a microcentrifuge tube, which was washed with 100. mu.l of solution D to recover any remaining spleen which was then aspirated with 5-10 additional 5cc times through a 22-gauge needle.

The samples were evenly distributed between two microcentrifuge tubes and the following solutions were added in order and mixed by inversion after each addition: 50 μ l 2M sodium acetate pH 4.0, 0.5ml water-saturated phenol (Feishell technologies, Pittsburgh Pa.), 100 μ l chloroform/isoamyl alcohol 49:1 (Feishell technologies, Pittsburgh Pa.). The solution was vortex mixed for 10 seconds and incubated on ice for 15 minutes. After centrifugation at 14krpm for 20 minutes at 2-8 ℃, the aqueous phase was transferred to a fresh tube. An equal volume of water saturated phenol chloroform isoamyl alcohol (50:49:1) was added and the tube was vortex mixed for 10 seconds. After 15 min incubation on ice, the samples were centrifuged at 2-8 ℃ for 20 min and the aqueous phase was transferred to a fresh tube and precipitated with an equal volume of isopropanol at-20 ℃ for a minimum of 30 min. After centrifugation at 14krpm for 20 minutes at 4 ℃, the supernatant was aspirated, the tube was briefly centrifuged, and all traces of liquid of the RNA pellet (pellet) were removed.

The RNA pellets were each dissolved in 300. mu.l of solution D, combined and precipitated with an equal volume of isopropanol at-20 ℃ for a minimum of 30 minutes. The samples were centrifuged at 14krpm for 20 minutes at 4 ℃, the supernatant aspirated as before, and the samples rinsed with 100 μ Ι of ice-cold 70% ethanol. The sample was again centrifuged at 14krpm for 20 minutes at 4 ℃, 70% ethanol solution was aspirated, and the RNA pellet was vacuum dried. The pellet was resuspended in 100. mu.l sterile diethylpyrocarbonate-treated water. The use of an absorbance of 1.0 corresponds to a concentration of 40

μ g/ml, concentration determined by A260. RNA was stored at-80 ℃.

Preparation of complementary DNA (cDNA)

Total RNA purified from mouse spleen as described above was used directly as template for cDNA preparation. RNA (50. mu.g) was diluted to 100. mu.L with sterile water and 10. mu.L of 130 ng/. mu.L oligo dT12 (synthesized on an Applied Biosystems model 392 DNA synthesizer) was added. The sample was heated at 70 ℃ for 10 minutes and then cooled on ice. mu.L of 5 × first strand buffer (Gibco/BRL, Gathersburg, Maryland) was added to ice along with 20. mu.L of 0.1M dithiothreitol (Gibco/BRL, Gathersburg, Maryland), 10. mu.L of 20mM deoxynucleoside triphosphate (dNTP, Baoling man, Indonepex, Indiana) and 10. mu.L of water. The samples were then incubated at 37 ℃ for 2 minutes. mu.L of reverse transcriptase (SuperscriptTMII, Gibco/BRL, Gethersburg, Md.) was added and incubation continued at 37 ℃ for 1 hour. The cDNA product was used directly in the Polymerase Chain Reaction (PCR).

Amplification of antibody genes by PCR

To amplify substantially all of the H and L chain genes using PCR, primers corresponding to substantially all of the disclosed sequences were selected. Since the amino-terminal nucleotide sequences of H and L contain considerable diversity, 33 oligonucleotides were synthesized to serve as 5 'primers for the H chain and 29 oligonucleotides were synthesized to serve as 5' primers for the κ L chain, as described in US 6,555,310. The constant region nucleotide sequence of each strand requires only one 3 'primer for the H strand and one 3' primer for the kappa L strand.

For each primer pair, a 50 μ L reaction was performed with the following components: 50 μmol 5 'primer, 50 μmol 3' primer, 0.25 μ L Taq DNA polymerase (5 units/. mu.L, Boehringer Mannheim, Indiana), 3 μ L cDNA (prepared as described above), 5 μ L2 mM dNTP, 5 μ L10X Taq DNA polymerase buffer containing MgCl2 (Boehringer Mannheim, Indiana), and H₂O to 50. mu.L. Amplification was performed using a geneamp (r)9600 thermal cycler (Perkin Elmer, foster, ca) according to the following thermal cycling program: 1 minute at 94 ℃; 30 cycles of 94 ℃ for 20 seconds; 30 seconds at 55 ℃; and 72 ℃ for 30 seconds; 6 minutes at 72 ℃; 4 ℃ is prepared.

The dsDNA product of the PCR process is then subjected to asymmetric PCR using only 3' primers to produce essentially only the antisense strand of the target gene. For each dsDNA product, a 100 μ Ι _ reaction was performed with the following composition: 200 μmol 3' primer, 2 μ L ds-DNA product, 0.5 μ L Taq DNA polymerase, 10 μ L2 mM dNTP, 10 μ L MgCl ₂10 Taq DNA polymerase buffer (precious man, indianapolis, indiana) and H₂O to 100. mu.L. The same PCR procedure as described above was used to amplify single stranded (ss) -DNA.

Purification of single stranded DNA and kinase activation of single stranded DNA by high performance liquid chromatography

The H chain ss-PCR product and L chain single stranded PCR product were ethanol precipitated by adding 2.5 volumes ethanol and 0.2 volumes of 7.5M ammonium acetate and incubated at-20 ℃ for at least 30 minutes. The DNA was pelleted by centrifugation in an Eppendorf centrifuge at 14krpm for 10 minutes at 2-8 ℃. The supernatant was carefully aspirated and the tube was centrifuged briefly again. Remove the last drop of supernatant with a pipette. The DNA was dried under vacuum at moderate temperature for 10 minutes. The H chain products were pooled in 210. mu.L water and the L chain products were pooled separately in 210. mu.L water. Single-stranded DNA was purified by High Performance Liquid Chromatography (HPLC) using Hewlett Packard 1090HPLC and Gen-PakTM FAX anion exchange column (Millipore Corp., Mirford, Mass.). The gradient used to purify single stranded DNA is shown in table 1, and the oven temperature is 60 ℃. The absorbance was monitored at 260 nm. Single-stranded DNA eluted by HPLC was collected in 0.5 minute fractions. Fractions containing single stranded DNA were ethanol precipitated, pooled and dried as described above. The dried DNA pellets were pooled in 200. mu.L of sterile water.

TABLE 1 HPLC gradients for ss-DNA purification

Time (minutes)	％A	％B	％C	Flow rate (ml/min)
					0	70	30	0	0.75
2	40	60	0	0.75
					17	15	85	0	0.75
18	0	100	0	0.75
					23	0	100	0	0.75
24	0	0	100	0.75
					28	0	0	100	0.75
29	0	100	0	0.75
					34	0	100	0	0.75
35	70	30	0	0.75

Buffer A was 25mM Tris, 1mM EDTA, pH8.0

Buffer B was 25mM Tris, 1mM EDTA, 1M NaCl, pH8.0

Buffer C was 40mm phosphoric acid

Single-stranded DNA was 5' -phosphorylated for mutagenesis at the time of preparation. mu.L of 10-kinase buffer (United States Biochemical, Cleveland, Ohio), 10.4. mu.L of 10mM adenosine-5' -triphosphate (Boehringer Mannheim, Indianapolis, Indiana) and 2. mu.L of polynucleotide kinase (30 units/. mu.L, U.S. Biochemical, Cleveland, Ohio) were added to each sample and the tubes were incubated at 37 ℃ for 1 hour. The reaction was terminated by incubating the tubes for 10 minutes at 70 ℃. The DNA was purified by 1 extraction with Tris equilibrated phenol (pH >8.0, Biochemical company, Cleveland, Ohio) chloroform isoamyl alcohol (50:49:1) and 1 extraction with chloroform isoamyl alcohol (49: 1). After extraction, the DNA was ethanol precipitated and concentrated as described above. The DNA aggregate was dried and then dissolved in 50. mu.L of sterile water. The concentration was determined by measuring the absorbance of a DNA aliquot at 260nm using 33. mu.g/ml corresponding to an absorbance of 1.0. Samples were stored at-20 ℃.

Preparation of uracil templates for use in generating spleen antibody phage libraries

1ml of E.coli CJ236 (Burley, Heracleus, Calif.) overnight culture was added to 50ml of 2 YT in 250ml baffled shake flasks. Cultures were grown at 37 ℃ to OD600 ═ 0.6, inoculated with 10 μ Ι 1/100 diluted stock of BS45 vector phage (described in US 6,555,310) and grown for a further 6 hours. Approximately 40ml of the culture was centrifuged at 12krpm for 15 minutes at 4 ℃. The supernatant (30ml) was transferred to fresh centrifuge tubes and incubated for 15 minutes at room temperature after addition of 15. mu.l of 10mg/ml RNase A (Boehringer Mannheim, Indianapolis, Ind). Phage were precipitated by adding 7.5ml of 20% polyethylene glycol 8000 (hehl scientific, pittsburgh, pennsylvania)/3.5M ammonium acetate (Sigma Chemical Co.), st louis, missouri) and incubating on ice for 30 minutes. The samples were centrifuged at 12krpm for 15 minutes at 2-8 ℃. The supernatant was carefully discarded and the tube was briefly centrifuged to remove all traces of supernatant. The pellet was resuspended in 400. mu.l of high salt buffer (300mM NaCl, 100mM Tris pH8.0, 1mM EDTA) and transferred to a 1.5ml tube.

Phage stocks were extracted repeatedly with equal volumes of equilibrated phenol chloroform isoamyl alcohol (50:49:1) until no trace of white interfaces were seen, and then extracted with equal volumes of chloroform isoamyl alcohol (49: 1). The DNA was precipitated with 2.5 volumes ethanol and 1/5 volumes of 7.5M ammonium acetate and incubated at-20 ℃ for 30 minutes. The DNA was centrifuged at 14krpm for 10 minutes at 4 ℃ and the pellet was washed 1 time with cold 70% ethanol and dried in vacuo. Uracil template DNA was dissolved in 30. mu.l of sterile water and the concentration was determined by A260 using an absorbance of 1.0 corresponding to a concentration of 40. mu.g/ml. The template was diluted to 250 ng/. mu.L with sterile water, aliquoted and stored at-20 ℃.

Mutagenesis of uracil templates with ss-DNA and electroporation into E.coli to generate antibody phage libraries

Antibody phage display libraries were generated by simultaneously introducing single chain heavy and light chain genes onto phage display vector uracil templates. Typical mutagenesis was performed on a2 μ g scale by mixing the following components in a 0.2ml PCR reaction tube in the following manner: mu.l (250 ng/. mu.L) uracil template, 8. mu.l 10 × annealing buffer (200mM Tris pH 7.0, 20mM MgCl2, 500mM NaCl), 3.33. mu.l kinase-activated single-stranded heavy chain insert (100 ng/. mu.L), 3.1. mu.l kinase-activated single-stranded light chain insert (100 ng/. mu.L) and sterile water to 80. mu.l. The DNA was annealed in a GeneAmp (R)9600 thermal cycler using the following thermal profile: at 94 ℃ for 20 seconds, 85 ℃ for 60 seconds, decreasing from 85 ℃ to 55 ℃ over 30 minutes, and holding at 55 ℃ for 15 minutes. After the procedure was completed, the DNA was transferred to ice. The extension/connection is performed by: mu.l of 10 × Synthesis buffer (5mM each dNTP, 10mM ATP, 100mM Tris pH 7.4, 50mM MgCl2, 20mM DTT), 8. mu. L T4 DNA ligase (1U/. mu.L, Boehringer, Indiana Polish), 8. mu.L diluted T7 DNA polymerase (1U/. mu.L, New England Biolabs, Beverly, Mass.) was added and incubated at 37 ℃ for 30 minutes. The reaction was stopped with 300. mu.l of mutagenesis stop buffer (10mM Tris pH8.0, 10mM EDTA). Mutagenized DNA was extracted once with equilibrated phenol (pH >8) chloroform isoamyl alcohol (50:49:1), once with chloroform isoamyl alcohol (49:1), and the DNA was ethanol precipitated at-20 ℃ for at least 30 minutes. The DNA was pelleted and the supernatant was carefully removed as described above. The sample was centrifuged again briefly and all traces of ethanol were removed with a pipette. The aggregate was dried under vacuum. The DNA was resuspended in 4. mu.l of sterile water.

Using electroporation, 1. mu.l of mutagenized DNA (500ng) was transferred to 40. mu.l of electrocompetent E.coli DH12S (Gibco/BRL Co., Gathersburg, Maryland). The transformed cells were mixed with approximately 1.0ml of overnight XL-1 cells diluted to 60% of the initial volume with 2 × YT broth. This mixture was then transferred to a 15ml sterile culture tube and 9ml of top agar was added for plating onto 150mm LB agar plates. The plates were incubated at 37 ℃ for 4 hours and then transferred to 20 ℃ overnight. First round antibody phages were made by eluting the phages from these plates with 10ml 2 YT, centrifuging to remove debris and taking the supernatant. These samples are antibody phage display libraries used to select antibodies against BST 1. By coating 10. mu.l of 10 on LB agar plates^-4The efficiency of electroporation was measured by suspending the cells in dilution, followed by incubating the plates overnight at 37 ℃. By combining 10^-4Number of plaques on dilution plate multiplied by 10⁶And calculating the efficiency. Under these conditions, the efficiency of library electroporation is typically greater than 1X 10⁷And (4) phage.

Transformation of E.coli by electroporation

Electrocompetent E.coli cells were thawed on ice. DNA was mixed with 40. mu.L of these cells by gently pumping the cells up and down 2-3 times, taking care not to introduce air bubbles. The cells were transferred to Gene Pulser cuvettes (0.2cm gap, Burley (BioRAD), Heracle, Calif.) that had been chilled on ice, again taking care not to introduce air bubbles at the time of transfer. The cuvettes were placed in an E.coli pulse generator (Burley, Heracles, Calif.) and electroporated according to the manufacturer's recommendations with a voltage set at 1.88 kV. The transformed samples were immediately resuspended in 1ml of 2 YT broth or 400 μ l2 YT/600 μ l overnight XL-1 cell mixture and processed as described.

Coating of M13 phage or cells transformed with antibody phage display vector mutagenesis reactions

Phage samples were added to 200. mu.L of E.coli XL1-Blue overnight culture (when plated on 100mm LB agar plates), or to 600. mu.L of overnight cells (when plated on 150mm plates in sterile 15ml culture tubes). After addition of LB top agar (3 ml for 100mm plates or 9ml for 150mm plates; top agar stored at 55 deg.C (see appendix A1, Sambrook et al, supra)), the mixture was distributed evenly on pre-heated (37 deg.C-55 deg.C) LB agar plates to remove any excess moisture on the agar surface. The plate was cooled at room temperature until the top agar solidified. Plates were left behind and incubated at 37 ℃ as indicated.

Preparation of biotinylated ADP-ribosyl cyclase 2 and biotinylated antibody

The concentrated recombinant BST1 antigen (full-length extracellular domain) was extensively dialyzed against BBS (20mM borate, 150mM NaCl, 0.1% NaN₃pH 8.0). After dialysis, 1mg of BST1(1mg/ml in BBS) was reacted with a 15-fold molar excess of biotin-XX-NHS ester (Molecular Probes, Eukin, Oreg., 40mM stock solution in DMSO). The reaction was incubated at room temperature for 90 minutes and then quenched with 20mM final concentration of taurine (sigma chemical, st louis, missouri). The biotinylation reaction mixture was then dialyzed against BBS at 2-8 ℃. After dialysis, biotinylated BST1 was diluted in panning buffer (40mM Tris, 150mM NaCl, 20mg/ml bsa, 0.1% tween 20, pH 7.5), aliquoted and stored at-80 ℃ until needed.

The antibody was reacted with 3- (N-maleimidopropanoyl) biotin (molecular probes, uk, oregon) using a free cysteine at the carboxy-terminus of the heavy chain. The antibody was reduced at room temperature for 30 minutes by adding DTT to a final concentration of 1 mM. The reduced antibody was passed through a Sephadex G50 desalting column equilibrated in 50mM potassium phosphate, 10mM boric acid, 150mM NaCl, pH 7.0. 3- (N-Maleimidopropionyl) -biotin was added to a final concentration of 1mM, and the reaction was allowed to proceed for 60 minutes at room temperature. The samples were then dialyzed well against BBS and stored at 2-8 ℃.

Preparation of avidin magnetic latex

The magnetic latex was thoroughly resuspended (Estapor, 10% solids, Bangs Laboratories, Inc., Poisson, Ind.) and aliquoted in 2ml aliquots into 15ml conical tubes. The magnetic latex was suspended in 12ml of distilled water and separated from the solution for 10 minutes using a magnet (PerSeptive Biosystems, Fremingham, Mass.). While maintaining separation of the magnetic latex with a magnet, the liquid was carefully removed using a 10ml sterile pipette. This washing process was repeated an additional 3 times. After the last wash, the latex was resuspended in 2ml of distilled water. In a separate 50ml conical tube, 10mg avidin-HS (NeutrAvidin, Pierce, Rockford, Ill.) was dissolved in 18ml40mM Tris, 0.15M sodium chloride, pH 7.5 (TBS). Under vortex mixing, 2ml of the washed magnetic latex was added to the diluted avidin-HS and the mixture was mixed for an additional 30 seconds. The mixture was incubated at 45 ℃ for 2 hours with shaking every 30 minutes. The avidin magnetic latex was separated from the solution using a magnet and washed 3 times with 20ml BBS as described above. After the last wash, the latex was resuspended in 10ml BBS and stored at 4 ℃.

Immediately prior to use, the avidin magnetic latex was equilibrated in panning buffer (40mM Tris, 150mM NaCl, 20mg/ml BSA, 0.1% Tween 20, pH 7.5). The avidin magnetic latex required for the panning experiment (200 μ l/sample) was added to a sterile 15ml centrifuge tube and made up to 10ml with panning buffer. The tube was placed on a magnet for 10 minutes to allow the latex to separate. The solution was carefully removed with a 10ml sterile pipette as described above. The magnetic latex was resuspended in 10ml of panning buffer to begin a second wash. The magnetic latex was washed with the panning buffer a total of 3 times. After the last wash, the latex was resuspended in panning buffer to the starting volume.

Example 2: selection of recombinant polyclonal antibodies against the BST1 antigen

Binding agents that specifically bind BST1 were selected from phage display libraries generated from hyperimmunized mice as described in example 1.

Panning (panning)

First round antibody phages were prepared using the BS45 uracil template as described in example 1. Electroporation of the mutagenized DNA was performed to generate phage samples derived from different immunized mice. To generate greater diversity in the recombinant polyclonal library, each phage sample was panned individually.

Phage displaying heavy and light chains on their surface in the antibody phage library were selected by panning with 7F 11-magnetic latex prior to a first round of functional panning with biotinylated BST1 antigen (as described in examples 21 and 22 of US 6,555,310). These enriched libraries were subjected to functional panning as described in example 16 of US 6,555,310 in principle. Specifically, 10. mu.L of 1X 10 was added^-6M biotinylated BST1 antigen was added to the phage sample (final concentration of approximately 1X 10 for BST 1)^-8M), and the mixture is allowed to equilibrate at 2-8 ℃ overnight.

After reaching equilibrium, the sample was panned with avidin magnetic latex to capture antibody phage that bound BST 1. The equilibrated avidin magnetic latex (example 1) (200. mu.L of latex per sample) was incubated with the phage for 10 minutes at room temperature. After 10 minutes, approximately 9ml of panning buffer was added to each phage sample and the magnetic latex was separated from the solution using a magnet. After 10 minutes of separation, unbound phage was carefully removed using a 10ml sterile pipette. The magnetic latex was then resuspended in 10ml of panning buffer to begin a second wash. The latex was washed a total of 3 times as described above. For each wash, the tube was contacted with the magnet for 10 minutes to allow unbound phage to separate from the magnetic latex. After the third wash, the magnetic latex was resuspended in 1mL of panning buffer and transferred to a 1.5mL tube. The entire volume of magnetic latex from each sample was then collected and resuspended in 200 μ l2 × YT and plated on 150mm LB plates as described in example 1 to amplify the bound phage. Plates were incubated at 37 ℃ for 4 hours, followed by overnight incubation at 20 ℃.

A150 mm plate used to amplify the bound phage was used to generate the next round of antibody phage. After overnight incubation, the second round of antibody phage was eluted from the 150mm plate by pumping 10mL of 2 × YT medium onto the lawn (lawn) and gently shaking the plate for 20 minutes at room temperature. The phage samples were then transferred to 15ml disposable sterile centrifuge tubes with stoppered caps and the debris from the LB plates was pooled by centrifuging the tubes at 3500rpm for 15 minutes. The supernatant containing the second round of antibody phage was then transferred to a new tube.

A second round of functional panning was established by diluting 100. mu.L of each phage stock solution into 900. mu.L of panning buffer in a 15ml disposable sterile centrifuge tube. Biotinylated BST1 antigen was then added to each sample as described in the first round of panning, and the phage samples were incubated at room temperature for 1 hour. Phage samples were then panned with avidin magnetic latex as described above. The course of panning was monitored at this point by plating aliquots of each latex sample on 100mm LB agar plates to determine the percentage of kappa positive. Most of the latex (99%) from each panning was plated on 150mm LB agar plates to amplify the latex-bound phage. 100mm LB agar plates were incubated at 37 ℃ for 6-7 hours, after which the plates were transferred to room temperature and nitrocellulose filters (pore size 0.45mm, BA85 Protran, Schleicher and Schuel corporation (Schleicher and Schuel), Schleicher Schuel, Schhann, N.H.) were overlaid onto the plaques.

Plates with nitrocellulose filters were incubated overnight at room temperature and then developed with goat anti-mouse kappa alkaline phosphatase conjugate to determine the percent kappa positive, as described below. Lower percentage of kappa positives in the population (<70%) phage samples were subjected to a round of panning using 7F 11-magnetic latex followed by approximately 2X 10^-9M biotinylated BST1 antigen was subjected to a third round of functional panning overnight at 2-8 ℃. Kappa positivity was also monitored for this round of panning. Individual phage samples with a kappa percent greater than 80% were pooled and subjected to a final round of panning at 2-8 ℃ and 5X 10-9M overnight. A wash from the fourth round of functional elutriationThe BST1 antibody gene contained in the removed phage is subcloned into expression vector pBRncoH 3.

In general, the subcloning process was performed as described in example 18 of US 6,555,310. After subcloning, the expression vector was electroporated into DH10B cells and the mixture was grown overnight in 2 × YT containing 1% glycerol and 10 μ g/ml tetracycline. After a second round of growth and selection in tetracycline, aliquots of the cells were frozen at-80 ℃ as a source for the production of BST1 polyclonal antibodies. Monoclonal antibodies were selected from these polyclonal mixtures by coating samples of the polyclonal mixture on LB agar plates containing 10. mu.g/ml tetracycline and screening for antibodies that recognize BST 1.

Expression and purification of recombinant antibodies against ADP-ribosyl cyclase 2

Shake flask inocula were generated from-70 ℃ cell banks overnight in Innova 4330 shaker (New Brunswick Scientific, Edison, N.J.) set at 37 ℃ and 300 rpm. The inoculum was used to inoculate a 20L fermentor (Applikon, Foster Calif.) containing a defined medium [ Pack et al (1993) Bio/Technology 11: 1271-. The temperature, pH and dissolved oxygen in the fermentor were controlled at 26 deg.C, 6.0-6.8 and 25% saturation, respectively. The foam was controlled by the addition of polypropylene glycol (Dow, midland, michigan). Glycerol was added to the fermentor in fed batch mode. Fab expression was induced by addition of L (+) -arabinose (Sigma, St. Louis, Mo.) to 2g/L during the late logarithmic growth phase. Cell density was measured by optical density at 600nm in a UV-1201 spectrophotometer (Shimadzu, Columbia, Md.). After the operation was terminated and the pH was adjusted to 6.0, the culture was passed through a M-210B-EH microfluidizer (Microfluidics, N.Marseilles) 2 times at 17,000 psi. High pressure homogenization of the cells released Fab into the culture supernatant.

The first step of purification is expanded bed immobilized metal affinity chromatography (EB-IMA)C) In that respect Mixing 0.1M NiCl₂Charging into Streamline^TMChelating resin (Pharmacia, Piscataway, N.) and then flowing it in the up-flow direction 50mM acetate, 200mM NaCl, 10mM imidazole, 0.01% NaN₃Swelling and equilibration in pH 6.0 buffer. Stock solution was used to homogenize the culture to 10mM imidazole, after which the culture homogenate was diluted two or more times in equilibration buffer to reduce the wet solids content to less than 5% by weight. The culture homogenate was then loaded onto a Streamline column flowing in the upstream direction at an apparent velocity of 300 cm/hr. Cell debris passes unhindered, but Fab is captured by high affinity interaction between nickel and the hexahistidine tag on the Fab heavy chain. After washing, the expanded bed was converted to a packed bed and flowed in the down-flow direction with 20mM borate, 150mM NaCl, 200mM imidazole, 0.01% NaN₃And pH8.0 buffer eluted Fab.

The second step of purification uses Ion Exchange Chromatography (IEC). Q Sepharose Fastflow resin (pharmacia, Piscataway, N.) in 20mM borate, 37.5mM NaCl, 0.01% NaN₃And pH 8.0. Fab elution pool from EB-IMAC step at 20mM borate, 0.01% NaN₃Diluted 4-fold in pH8.0 and loaded onto an IEC column. After washing, the Fab was eluted with a salt gradient of 37.5-200mM NaCl. Before pooling, Xcell II was used^TMThe purity of the eluted fractions was assessed by SDS-PAGE system (Novex Corp., san Diego, Calif.). Finally, the Fab pool was concentrated and diafiltered to 20mM borate, 150mM NaCl, 0.01% NaN₃pH8.0 buffer for storage. Then at a Sartocon Slice equipped with a 10,000MWCO cassette^TMImplemented in a system (Sartorius, boschidia, new york). The final purification yield is typically 50%. The concentration of the purified Fab was measured by UV absorbance at 280nm, assuming an absorbance of 1.6 corresponding to a 1mg/ml solution.

Example 3: specificity of monoclonal antibodies to BST1 as determined by flow cytometry

The specificity of the antibodies against BST1 selected in example 2 was tested by flow cytometry. To test the ability of the antibodies to bind to cell surface BST1 protein, the antibodies were incubated with cells a549 and H226 expressing BST1 from human lung adenocarcinoma and human lung squamous carcinoma, respectively. Cells were washed in FACS buffer (DPBS, 2% FBS), centrifuged and resuspended in 100 μ l of diluted BST1 primary antibody (also diluted in FACS buffer). The antibody-a 549 complex was incubated on ice for 60 minutes and then washed twice with FACS buffer as described above. Cell-antibody pellets were resuspended in 100 μ l of diluted secondary antibody (also diluted in FACS buffer) and incubated on ice for 60 minutes. The pellet was washed as before and resuspended in 200. mu.l FACS buffer. Samples were loaded onto a BD facscan to II flow cytometer and data was analyzed using BDFACSdiva software.

Results

The results of flow cytometry analysis showed that 4 monoclonal antibodies, designated BST1_ a1, BST1_ a2, and BST _ A3, efficiently bound to cell surface human BST 1. Figure 3a shows the binding specificity of both BST1_ a1 and BST1_ a2 to BST1 on a549 and H226 cells, respectively. Figure 3b shows the binding specificity of BST1_ A3 to BST1 on a549 and H226 cells. The results show strong binding of these antibodies to BST1 on a549 and H226.

Example 4: structural characterization of monoclonal antibodies against BST1

The cDNA sequences encoding the heavy and light chain variable regions of the BST1_ a2 and BST1_ a1 monoclonal antibodies were obtained using standard PCR techniques and sequenced using standard DNA sequencing techniques.

Antibody sequences may be mutagenized to back-mutate to germline residues at one or more residues.

The nucleotide and amino acid sequences of the heavy chain variable region of BST1_ A2 are SEQ ID NOs 6 and 2, respectively.

The nucleotide and amino acid sequences of the light chain variable region of BST1_ A2 are SEQ ID NOs: 8 and 4, respectively.

The nucleotide and amino acid sequences of the heavy chain of BST _ A2 are SEQ ID NOS: 73 and 74, respectively.

The nucleotide and amino acid sequences of the light chain of BST _ A2 are SEQ ID NOS: 75 and 76, respectively.

BST1_ A2 heavy chain immunoglobulin sequence and its knownComparison of murine germline immunoglobulin heavy chain sequences shows that the BST1_ A2 heavy chain utilizes light from murine germline V_H1-39. Further analysis of BST1_ A2V Using the Kabat System of CDR region determination_HThe sequences delineate the heavy chain CDR1, CDR2 and CDR3 regions, as shown in SEQ ID NOs 10, 12 and 14, respectively. BST1_ A2CDR1 and CDR 2V are shown in FIGS. 1a and 1b_HSequences and germline V_H1-39 sequence alignment.

Comparison of the BST1_ a2 light chain immunoglobulin sequence with the known murine germline immunoglobulin light chain sequence shows that the BST1_ a2 light chain utilizes light chains from murine germline V_KV of 4 to 55_KAnd (4) a section. Further analysis of BST1_ A2V Using the Kabat System of CDR region determination_KThe sequences circumscribe the light chain CDR1, CDR2, and CDR3 regions as shown in SEQ ID NOs 16, 18, and 20, respectively. Alignment of BST1_ A2CDR1, CDR2 and CDR3 VK sequences to germline VK 4-55 sequences is shown in fig. 2a, 2b and 2 c.

The nucleotide and amino acid sequences of the heavy chain variable region of BST1_ A1 are SEQ ID NOs: 5 and 1, respectively.

The nucleotide and amino acid sequences of the light chain variable region of BST1_ A1 are SEQ ID NOs 7 and 3, respectively.

Comparison of the BST1_ A1 heavy chain immunoglobulin sequence with known murine germline immunoglobulin heavy chain sequences shows that the BST1_ A1 heavy chain utilizes heavy chains from murine germline V_HV of 1 to 80_HAnd (4) a section. Further analysis of BST1_ A1V Using the Kabat System of CDR region determination_HThe sequences delineate the heavy chain CDR1, CDR2 and CDR3 regions, as shown in SEQ ID NOs 9, 11 and 13, respectively. In FIGS. 1a and 1b, BST1_ A1CDR1 and CDR 2V are shown_HSequences and germline V_H1-80 sequence alignment.

Comparison of the BST1_ a1 light chain immunoglobulin sequence with the known murine germline immunoglobulin light chain sequence shows that the BST1_ a1 light chain utilizes light chains from murine germline V_KV of 4 to 74_KAnd (4) a section. Further analysis of the BST1_ A1 VK sequence using the Kabat system of CDR region determination circumscribed a light chain CDR1 region, a CDR2 region, and a CDR3 region as set forth in SEQ ID NOS: 15, 17, and 19, respectively. Alignment of BST1_ a1CDR1, CDR2 and CDR3 VK sequences with germline VK 4-74 sequences is shown in fig. 2a, 2b and 2 c.

The nucleotide and amino acid sequences of the heavy chain variable region of BST1_ A3 are SEQ ID NOs 54 and 52, respectively.

The nucleotide and amino acid sequences of the light chain variable region of BST1_ A3 are SEQ ID NOs: 55 and 53, respectively.

Comparison of the BST1_ A3 heavy chain immunoglobulin sequence with known murine germline immunoglobulin heavy chain sequences shows that the BST1_ A3 heavy chain utilizes light from murine V_HV of germline 69-1_HAnd (4) a section. Further analysis of BST1_ A3V Using the Kabat System of CDR region determination_HThe sequences delineate the heavy chain CDR1, CDR2, and CDR3 regions, as shown in SEQ ID NOs 56, 57, and 58, respectively. In FIGS. 1a and 1b, BST1_ A3CDR1 and CDR 2V are shown_HSequence and mouse V_HAlignment of germline 69-1 sequences.

Comparison of the BST1_ A3 light chain immunoglobulin sequence with the known murine germline immunoglobulin light chain sequence shows that the BST1_ A3 light chain utilizes light chains from murine V_KV of germline 44-1_KAnd (4) a section. Further analysis of BST1_ A3V Using the Kabat System of CDR region determination_KThe sequences delineate the light chain CDR1, CDR2, and CDR3 regions, as shown in SEQ ID NOs 59, 60, and 61, respectively. In FIGS. 2a, 2b and 2c BST1_ A3CDR1, CDR2 and CDR 3V are shown_KSequence and mouse V_KAlignment of germline 44-1 sequences.

Example 5: internalization of BST1_ a1 and BST1_ a2 in a549 and H226 cells and MabZAP

Internalization of BST1_ a1 and BST1_ a2 by H226 and a549 was studied using the MabZap assay. The MabZAP assay showed that the anti-BST 1 monoclonal antibody was internalized by binding to an anti-human IgG secondary antibody conjugated to the toxin saporin (saporin). (Advanced Targeting System, IT-22-100, san Diego, Calif.). First, BST1 Fab was bound to the surface of the cells. Subsequently, MabZAP antibodies bind to the primary antibody. Subsequently, the MabZAP complex is internalized by the cell. Saporin enters cells, resulting in inhibition of protein synthesis and ultimately cell death.

The MabZAP test was performed as follows. The cells were cultured at 5X 10³Density of individual cells/well. anti-BST 1 monoclonal antibody or isotype control human IgG was serially diluted, then added to cells and incubated at 25 ℃For 15 minutes. MabZAP was then added and incubated for 72 hours at 37 ℃. By CellTiter-

Luminescence cell viability Assay (Luminescent cellviatility Assay) kit (Promega, G7571) detects cell viability in the plates and the plates are read and analyzed using Promega Glomax. Cell death is directly proportional to the concentration of anti-BST 1 monoclonal antibody. Figures 4a and 4b show that the anti-BST 1 monoclonal antibodies BST1_ a1 and BST1_ a2 are efficiently internalized by H226 and a549 cells compared to anti-human IgG isotype control antibodies.

Example 6: humanization of BST 1A 2

To design BST1_ A2V_HAnd V_LThe humanized sequence of (1), identifying framework amino acids important for the formation of the CDR structure using a three-dimensional model. Human V with high homology to BST1_ A2 was also selected from GenBank database_HAnd V_LAnd (4) sequencing. The CDR sequences, along with the identified framework amino acid residues, were grafted from BST1_ a2 to human framework sequences (fig. 5-7).

Example 7: anti-BST 1 mAbs mediated antibody-dependent cytotoxicity

First, 25 μ L of parental and non-fucosylated anti-BST 1 antibodies (BST1_ a2 and BST1_ a2_ NF) were added to individual wells of a v-bottom 96-well plate at a concentration of 10 nm/L to 0.1 nm/L, and 50 μ L of BST 1-expressing a549 and U937 cells. Subsequently 25 μ l of effector cells were added to the wells to generate final effector to target (E: T) ratios of 10:1 and 25: 1. The plates were then gently centrifuged at 1000 rpm for 2 minutes, after which they were incubated at 37 ℃ in a 5% CO2 incubator for 4 hours. At 3 hours after incubation, 10 μ Ι lysis solution was added to each well containing only cells expressing BST1 to measure the maximum LDH release, and to a set of wells containing only medium for volume corrected control.

After incubation, cells were gently centrifuged at 1000 rpm for 2 minutes, after which 50 μ Ι of supernatant was transferred to a flat bottom 96 well plate. Using Cyto available from Promega

Non-radioactive cytotoxicity assay (catalog No. G1780), kit components were reconstituted according to the manufacturer's instructions and 50. mu.l of substrate mixture was then added to each well. Plates were then covered and incubated for 30 minutes at 25 ℃ in the dark. Thereafter, 50. mu.l of stop solution was added to each well and the absorbance at 490 nm was recorded using a varioskan plate microplate reader.

Using an antibody known to stimulate cell killing via ADCC as a positive control and using a human IgG1 isotype control as a negative control, the results show: BST1_ a2 and BST1_ a2_ NF are able to elicit ADCC on a549 and U937 cells expressing BST 1. On a549 cells expressing BST1, BST1_ a2_ NF has been shown to have about 45% killing at 10nmol/L (fig. 8 a). On U937 cells expressing BST1, BST1_ A2 has been shown to have about 20% killing at 1nmol/L, and BST1_ A2_ NF has been shown to have about 45% killing at 1nmol/L (FIG. 8 b).

Example 8: specificity of monoclonal antibodies to BST1 in AML patients as determined by flow cytometry

BST _ a2 was tested for its ability to bind to lymphoblasts from AML patients by flow cytometry. Blood was obtained from 20 AML patients. Using the procedure as described in example 3, BST _ a2 has been shown to bind to AML blasts in approximately 80% of AML patients.

Example 9: ADCC induced by BST1_ A2 antibody and 5-azacytidine (AZA)

K052 cells (AML cell line Fab M2) and PBMCs were pretreated with 0.5 or 0.1 μ M5-azacytidine (AZA) for 48 and 24 hours, respectively, and then incubated with a tenfold dilution of BST1_ a2 antibody (10 to 0.01 μ g/ml) for 4 hours. LDH release was measured to detect cell lysis (Promega kit). The results are shown in fig. 9. The graph represents the ADCC percentage in the presence of BST1_ a2 antibody, either alone or in combination with AZA.

SKNO1 cells (AML cell line Fab M2) and PBMCs were pretreated with 2 or 0.5 μ M5-azacytidine (AZA) for 48 and 24 hours, respectively, and then incubated with a tenfold dilution of BST1_ a2 antibody (10 to 0.01 μ g/ml) for 4 hours. LDH release was measured to detect cell lysis (Promega kit). The results are shown in fig. 10. The graph represents the ADCC percentage in the presence of BST1_ a2 antibody, either alone or in combination with AZA.

The following table shows the combination indices calculated using the Chou and Talalay method (Calcusyn software) at different combination ratios between the BST1_ A2 antibody and 5-azacytidine.

(CI < 1: synergism; CI ═ 1: additive effects; CI > 1: antagonism). A high level of synergy is shown.

Example 10: ADCC induced by BST1_ A2 antibody and Decitabine (DEC)

SKNO1 cells (AML cell line Fab M2) and PBMCs were pretreated with 2 or 0.5 μ M Decitabine (DEC) for 48 and 24 hours, respectively, and then incubated with a tenfold dilution of BST1_ a2 antibody (10 to 0.01 μ g/ml) for 4 hours. LDH release was measured to detect cell lysis (Promega kit). The results are shown in fig. 11. The graph represents the ADCC percentage in the presence of BST1_ a2, either separately or in combination with DEC.

HL-60 cells (AML cell line Fab M2/M3) and PBMCs were pre-treated with 0.5 or 2 μ M Decitabine (DEC) for 48 and 24 hours, respectively, and then incubated with a tenfold dilution of BST1_ A2 antibody (10 to 0.01 μ g/ml) for 4 hours. LDH release was measured to detect cell lysis (Promega kit). The results are shown in fig. 12. The graph represents the ADCC percentage in the presence of BST1_ a2 antibody, either alone or in combination with DEC.

The following table shows the combination index calculated using the Chou and Talalay method (Calcusyn software) at different combination ratios between BST1_ a2 antibody and decitabine.

(CI < 1: synergism; CI ═ 1: additive effects; CI > 1: antagonism). A high level of synergy is shown.

Sequence of

Sequence listing

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Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

195 200 205

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

210 215 220

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Ser

225 230 235

<210>5

<211>811

<212>DNA

<213> mouse

<220>

<221>misc_feature

<222>(808)..(808)

<223> n is a, c, g or t

<400>5

acgctttgta catggagaaa ataaagtgaa acaaagcact attgcactgg cactcttacc 60

gctcttattt acccctgtgg caaaagccca ggtgaagctt cagcagtccg gggctgagct 120

ggtgaggcct gggtcctcag tgaagatttc ctgcaaggct tctggctacg cattcagtaa 180

ctcctggata aactgggtga agcagaggcc tggacagggt cttgagtgga ttggacagat 240

ttatcctgga gattatgata ctaactacaa tggaaaattc aagggtaaag ccacactgac 300

tgcagactac tcctccagca cagcctacat gcagctcaac agcctaacat ctgaggactc 360

tgcggtctat ttctgtgcaa gggggggatc gatctactat ggtaacctcg ggttcttcga 420

tgtctggggc gcagggacca cggtcaccgt ctcctcagcc aaaacgacac ccccatctgt 480

ctatccactg gcccctggat ctgctgccca aactaactcc atggtgaccc tgggatgcct 540

ggtcaagggc tatttccctg agccagtgac agtgacctgg aactctggat ccctgtccag 600

cggtgtgcac accttcccag ctgtcctgca gtctgacctc tacactctga gcagctcagt 660

gactgtcccc tccagcacct ggcccagcga gaccgtcacc tgcaacgttg cccacccggc 720

cagcagcacc aaggtggaca agaaaattgt gcccagggat tgtcatcatc accatcacca 780

tcactaattg acagcttatc atcgatangc t 811

<210>6

<211>803

<212>DNA

<213> mouse

<400>6

aaaaccctgg cgttacccac gctttgtaca tggagaaaat aaagtgaaac aaagcactat 60

tgcactggca ctcttaccgc tcttatttac ccctgtggca aaagcccagg cttatctaca 120

gcagtctgga cctgagctgg tgaaggctgg cgcttcagtg aagatgtcct gcaaggcttc 180

tggttactca ttcattgagt acaccataaa ctgggtgaaa cagagccatg gaaagagcct 240

tgagtggatt ggaaatattg atccttatta tggaaccact tattacaatc agatgttcac 300

gggcaaggcc acattgactg tagaccaatc ttccaacact gcctacatgc agctcaagag 360

cctgacatct gaggactctg cagtctattt ctgtgcaaga ggctccgcct ggtttcctta420

ctggggccag gggactctag tcactgtctc tgcagccaaa acgacacccc catctgtcta 480

tccactggcc cctggatctg ctgcccaaac taactccatg gtgaccctgg gatgcctggt 540

caagggctat ttccctgagc cagtgacagt gacctggaac tctggatccc tgtccagcgg 600

tgtgcacacc ttcccagctg tcctgcagtc tgacctctac actctgagca gctcagtgac 660

tgtcccctcc agcacctggc ccagcgagac cgtcacctgc aacgttgccc acccggccag 720

cagcaccaag gtggacaaga aaattgtgcc cagggattgt catcatcacc atcaccatca 780

ctaattgaca gcttatcatc gat 803

<210>7

<211>759

<212>DNA

<213> mouse

<400>7

gtttttttgg atggagtgaa acgatgaaat acctattgcc tacggcagcc gctggattgt 60

tattactcgc tgcccaacca gccatggccg aaatggttct cacccagtct ccagcaatca 120

tgtctacatc tctaggggaa cgggtcacca tgacctgcac tgccagctca cgtgtaagtt 180

ccagttactt gcactggtac cagcagaagc caggatcctc ccccaaactc tggatttata 240

gtacatccaa cctggcttct ggagtcccag ctcgcttcag tggcagtggg tctgggacct 300

cttactctct cacaatcagc agcatggagg ctgaagatgc tgccacttat tactgccacc 360

agtatcatcg ttccccgtac acgttcggag gggggaccaa gctggaaata aaacgggctg 420

atgctgcacc aactgtatcc atcttcccac catccagtga gcagttaaca tctggaggtg 480

cctcagtcgt gtgcttcttg aacaacttct accccaaaga catcaatgtc aagtggaaga 540

ttgatggcag tgaacgacaa aatggcgtcc tgaacagttg gactgatcag gacagcaaag 600

acagcaccta cagcatgagc agcaccctca cgttgaccaa ggacgagtat gaacgacata 660

acagctatac ctgtgaggcc actcacaaga catcaacttc acccattgtc aagagcttca 720

acaggaatga gtcttaagtg attagctaat tctagaacg 759

<210>8

<211>804

<212>DNA

<213> mouse

<400>8

actctctact gtttctccat acccgttttt ttggatggag tgaaacgatg aaatacctat 60

tgcctacggc agccgctgga ttgttattac tcgctgccca accagccatg gccgacatcg 120

ttatgtctca gtctccagca atcatgtctg catctccagg ggagaaggtc accatgacct 180

gcagtgccag ctcaagtgta acttacatgt actggtacca gcagaagcca ggatcctccc 240

ccagactcct gatttatgac acatccaacc tggcttctgg agtccctgtt cgcttcagtg 300

gcagtgggtc tgggacctct tactctctca caatcagccg aatggaggct gaagatactg 360

ccacttatta ctgccagcag tggagtaatt acccactcac gttcggtgct gggaccaagc 420

tggagctgaa acgggctgat gctgcaccaa ctgtatccat cttcccacca tccagtgagc 480

agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac cccaaagaca 540

tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg aacagttgga 600

ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg ttgaccaagg 660

acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca tcaacttcac 720

ccattgtcaa gagcttcaac aggaatgagt cttaagtgat tagctaattc tagaacgcgt 780

cacttggcac tggccgtcgt ttta 804

<210>9

<211>11

<212>PRT

<213> mouse

<400>9

Gly Tyr Ala Phe Ser Asn Ser Trp Ile Asn Trp

1 5 10

<210>10

<211>11

<212>PRT

<213> mouse

<400>10

Gly Tyr Ser Phe Ile Glu Tyr Thr Ile Asn Trp

1 5 10

<210>11

<211>17

<212>PRT

<213> mouse

<400>11

Gly Gln Ile Tyr Pro Gly Asp Tyr Asp Thr Asn Tyr Asn Gly Lys Phe

1 5 10 15

Lys

<210>12

<211>17

<212>PRT

<213> mouse

<400>12

Gly Asn Ile Asp Pro Tyr Tyr Gly Thr Thr Tyr Tyr AsnGln Met Phe

1 5 10 15

Thr

<210>13

<211>16

<212>PRT

<213> mouse

<400>13

Ala Arg Gly Gly Ser Ile Tyr Tyr Gly Asn Leu Gly Phe Phe Asp Val

1 5 10 15

<210>14

<211>9

<212>PRT

<213> mouse

<400>14

Ala Arg Gly Ser Ala Trp Phe Pro Tyr

1 5

<210>15

<211>12

<212>PRT

<213> mouse

<400>15

Thr Ala Ser Ser Arg Val Ser Ser Ser Tyr Leu His

1 5 10

<210>16

<211>10

<212>PRT

<213> mouse

<400>16

Ser Ala Ser Ser Ser Val Thr Tyr Met Tyr

1 5 10

<210>17

<211>7

<212>PRT

<213> mouse

<400>17

Ser Thr Ser Asn Leu Ala Ser

1 5

<210>18

<211>7

<212>PRT

<213> mouse

<400>18

Asp Thr Ser Asn Leu Ala Ser

1 5

<210>19

<211>9

<212>PRT

<213> mouse

<400>19

His Gln Tyr His Arg Ser Pro Tyr Thr

1 5

<210>20

<211>9

<212>PRT

<213> mouse

<400>20

Gln Gln Trp Ser Asn Tyr Pro Leu Thr

1 5

<210>21

<211>33

<212>DNA

<213> mouse

<400>21

ggctacgcat tcagtaactc ctggataaac tgg 33

<210>22

<211>33

<212>DNA

<213> mouse

<400>22

ggttactcat tcattgagta caccataaac tgg 33

<210>23

<211>51

<212>DNA

<213> mouse

<400>23

ggacagattt atcctggaga ttatgatact aactacaatg gaaaattcaa g 51

<210>24

<211>51

<212>DNA

<213> mouse

<400>24

ggaaatattg atccttatta tggaaccact tattacaatc agatgttcac g 51

<210>25

<211>48

<212>DNA

<213> mouse

<400>25

gcaagggggg gatcgatcta ctatggtaac ctcgggttct tcgatgtc 48

<210>26

<211>27

<212>DNA

<213> mouse

<400>26

gcaagaggct ccgcctggtt tccttac 27

<210>27

<211>36

<212>DNA

<213> mouse

<400>27

actgccagct cacgtgtaag ttccagttac ttgcac 36

<210>28

<211>30

<212>DNA

<213> mouse

<400>28

agtgccagct caagtgtaac ttacatgtac 30

<210>29

<211>21

<212>DNA

<213> mouse

<400>29

agtacatcca acctggcttc t 21

<210>30

<211>21

<212>DNA

<213> mouse

<400>30

gacacatcca acctggcttc t 21

<210>31

<211>27

<212>DNA

<213> mouse

<400>31

caccagtatc atcgttcccc gtacacg 27

<210>32

<211>27

<212>DNA

<213> mouse

<400>32

cagcagtgga gtaattaccc actcacg 27

<210>33

<211>33

<212>DNA

<213> mouse

<400>33

ggctacgcat tcagtagcta ctggatgaac tgg 33

<210>34

<211>51

<212>DNA

<213> mouse

<400>34

ggacagattt atcctggaga tggtgatact aactacaacg gaaagttcaa g 51

<210>35

<211>33

<212>DNA

<213> mouse

<400>35

ggttactcat tcactgacta caacatgaac tgg 33

<210>36

<211>51

<212>DNA

<213> mouse

<400>36

ggagtaatta atcctaacta tggtactact agctacaatc agaagttcaa g 51

<210>37

<211>36

<212>DNA

<213> mouse

<400>37

actgccagct caagtgtaag ttccagttac ttgcac 36

<210>38

<211>21

<212>DNA

<213> mouse

<400>38

agcacatcca acctggcttc t 21

<210>39

<211>25

<212>DNA

<213> mouse

<400>39

caccagtatc atcgttcccc accca 25

<210>40

<211>30

<212>DNA

<213> mouse

<400>40

agtgccagct caagtgtaag ttacatgtac 30

<210>41

<211>21

<212>DNA

<213> mouse

<400>41

gacacatcca acctggcttc t 21

<210>42

<211>25

<212>DNA

<213> mouse

<400>42

cagcagtgga gtagttaccc accca 25

<210>43

<211>318

<212>PRT

<213> Intelligent (Homo sapiens)

<400>43

Met Ala Ala Gln Gly Cys Ala Ala Ser Arg Leu Leu Gln Leu Leu Leu

1 5 10 15

Gln Leu Leu Leu Leu Leu Leu Leu Leu Ala Ala Gly Gly Ala Arg Ala

20 25 30

Arg Trp Arg Gly Glu Gly Thr Ser Ala His Leu Arg Asp Ile Phe Leu

35 40 45

Gly Arg Cys Ala Glu Tyr Arg Ala Leu Leu Ser Pro Glu Gln Arg Asn

50 55 60

Lys Asn Cys Thr Ala Ile Trp Glu Ala Phe Lys Val Ala Leu Asp Lys

65 70 75 80

Asp Pro Cys Ser Val Leu Pro Ser Asp Tyr Asp Leu Phe Ile Asn Leu

85 90 95

Ser Arg His Ser Ile Pro Arg Asp Lys Ser Leu Phe Trp Glu Asn Ser

100 105 110

His Leu Leu Val Asn Ser Phe Ala Asp Asn Thr Arg Arg Phe Met Pro

115 120 125

Leu Ser Asp Val Leu Tyr Gly Arg Val Ala Asp Phe Leu Ser Trp Cys

130 135 140

Arg Gln Lys Asn Asp Ser Gly Leu Asp Tyr Gln Ser Cys Pro Thr Ser

145 150 155 160

Glu Asp Cys Glu Asn Asn Pro Val Asp Ser Phe Trp Lys Arg Ala Ser

165 170 175

Ile Gln Tyr Ser Lys Asp Ser Ser Gly Val Ile His Val Met Leu Asn

180 185 190

Gly Ser Glu Pro Thr Gly Ala Tyr Pro Ile Lys Gly Phe Phe Ala Asp

195 200 205

Tyr Glu Ile Pro Asn Leu Gln Lys Glu Lys Ile Thr Arg Ile Glu Ile

210 215 220

Trp Val Met His Glu Ile Gly Gly Pro Asn Val Glu Ser Cys Gly Glu

225 230 235 240

Gly Ser Met Lys Val Leu Glu Lys Arg Leu Lys Asp Met Gly Phe Gln

245 250 255

Tyr Ser Cys Ile Asn Asp Tyr Arg Pro Val Lys Leu Leu Gln Cys Val

260 265 270

Asp His Ser Thr His Pro Asp Cys Ala Leu Lys Ser Ala Ala Ala Ala

275 280 285

Thr Gln Arg Lys Ala Pro Ser Leu Tyr Thr Glu Gln Arg Ala Gly Leu

290 295 300

Ile Ile Pro Leu Phe Leu Val Leu Ala Ser Arg Thr Gln Leu

305 310 315

<210>44

<211>264

<212>PRT

<213> Intelligent people

<400>44

Gly Ala Arg Ala Arg Trp Arg Gly Glu Gly Thr Ser Ala His Leu Arg

1 5 10 15

Asp Ile Phe Leu Gly Arg Cys Ala Glu Tyr Arg Ala Leu Leu Ser Pro

20 25 30

Glu Gln Arg Asn Lys Asn Cys Thr Ala Ile Trp Glu Ala Phe Lys Val

35 40 45

Ala Leu Asp Lys Asp Pro Cys Ser Val Leu Pro Ser Asp Tyr Asp Leu

50 55 60

Phe Ile Asn Leu Ser Arg His Ser Ile Pro Arg Asp Lys Ser Leu Phe

65 70 75 80

Trp Glu Asn Ser His Leu Leu Val Asn Ser Phe Ala Asp Asn Thr Arg

85 90 95

Arg Phe Met Pro Leu Ser Asp Val Leu Tyr Gly Arg Val Ala Asp Phe

100 105 110

Leu Ser Trp Cys Arg Gln Lys Asn Asp Ser Gly Leu Asp Tyr Gln Ser

115 120 125

Cys Pro Thr Ser Glu Asp Cys Glu Asn Asn Pro Val Asp Ser Phe Trp

130 135140

Lys Arg Ala Ser Ile Gln Tyr Ser Lys Asp Ser Ser Gly Val Ile His

145 150 155 160

Val Met Leu Asn Gly Ser Glu Pro Thr Gly Ala Tyr Pro Ile Lys Gly

165 170 175

Phe Phe Ala Asp Tyr Glu Ile Pro Asn Leu Gln Lys Glu Lys Ile Thr

180 185 190

Arg Ile Glu Ile Trp Val Met His Glu Ile Gly Gly Pro Asn Val Glu

195 200 205

Ser Cys Gly Glu Gly Ser Met Lys Val Leu Glu Lys Arg Leu Lys Asp

210 215 220

Met Gly Phe Gln Tyr Ser Cys Ile Asn Asp Tyr Arg Pro Val Lys Leu

225 230 235 240

Leu Gln Cys Val Asp His Ser Thr His Pro Asp Cys Ala Leu Lys Ser

245 250 255

Ala Ala Ala Ala Thr Gln Arg Lys

260

<210>45

<211>116

<212>PRT

<213> mouse

<400>45

Gln Ala Tyr Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Ala Gly Ala

1 510 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ile Glu Tyr

20 25 30

Thr Ile Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asp Pro Tyr Tyr Gly Thr Thr Tyr Tyr Asn Gln Met Phe

50 55 60

Thr Gly Lys Ala Thr Leu Thr Val Asp Gln Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Ser Ala Trp Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ala

115

<210>46

<211>116

<212>PRT

<213> Intelligent people

<400>46

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ile Glu Tyr

20 2530

Thr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asp Pro Tyr Tyr Gly Thr Thr Tyr Tyr Asn Gln Met Phe

50 55 60

Thr Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser Ala Trp Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>47

<211>117

<212>PRT

<213> Intelligent people

<220>

<221>misc_feature

<222>(31)..(35)

<223> Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(50)..(66)

<223> Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(99)..(106)

<223> Xaa can be any naturally occurring amino acid

<400>47

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Xaa Xaa

20 25 30

Xaa Xaa Xaa Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Arg Val Thr Leu Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Gly Gln Gly Thr Leu

100 105 110

Val Pro Val Ser Ser

115

<210>48

<211>106

<212>PRT

<213> mouse

<400>48

Asp Ile Val Met Ser Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Thr Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Thr Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Asn Tyr Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210>49

<211>106

<212>PRT

<213> Intelligent people

<400>49

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Thr Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Asn Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>50

<211>107

<212>PRT

<213> Intelligent people

<220>

<221>misc_feature

<222>(24)..(34)

<223> Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(50)..(56)

<223> Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(89)..(97)

<223> Xaa can be any naturally occurring amino acid

<400>50

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

20 25 30

Xaa Xaa Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

85 90 95

Xaa Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>51

<211>16

<212>PRT

<213> Intelligent people

<400>51

Asn Ile Asp Pro Tyr Tyr Gly Thr Thr Tyr Tyr Asn Gln Met Phe Gln

1 5 10 15

<210>52

<211>245

<212>PRT

<213> mouse

<400>52

Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr

1 5 10 15

Pro Val Ala Lys Ala Gln Val Gln Leu Gln Gln Ser Arg Ala Glu Leu

20 25 30

Val Met Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr

35 40 45

Thr Phe Ser Asp Tyr Trp Val His Trp Val Arg Gln Arg Pro Gly Gln

50 55 60

Gly Leu Glu Trp Ile Gly Ala Ile Asp Gly Ser Asp Thr Phe Asn Asp

65 70 75 80

Tyr Ser Gln Lys Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Glu Ser

85 90 95

Ser Ser Thr Val Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Gly Gly Leu Leu Gln Tyr Trp Gly Gln

115 120 125

Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val

130135 140

Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr

145 150 155 160

Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr

165 170 175

Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val

180 185 190

Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser

195 200 205

Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala

210 215 220

Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys His His

225 230 235 240

His His His His His

245

<210>53

<211>236

<212>PRT

<213> mouse

<400>53

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser

2025 30

Leu Ser Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Glu Asn Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys

50 55 60

Ser Pro Gln Leu Leu Val Tyr Asn Thr Lys Thr Leu Gly Glu Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys

85 90 95

Ile Asn Ser Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His

100 105 110

His Tyr Gly Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

115 120 125

Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser

130 135 140

Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn

145 150 155 160

Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu

165 170 175

Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp

180185 190

Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr

195 200 205

Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr

210 215 220

Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Ser

225 230 235

<210>54

<211>738

<212>DNA

<213> mouse

<400>54

gtgaaacaaa gcactattgc actggcactc ttaccgctct tatttacccc tgtggcaaaa 60

gcccaggtcc agctgcagca gtctagggct gaacttgtga tgcctggggc ttcagtgaag 120

atgtcctgca agacttctgg ctacacattc tctgactact gggtacactg ggtgaggcag 180

aggcctggac aaggccttga gtggatcgga gcgattgatg gttctgatac ttttaatgac 240

tacagtcaga agtttaaggg cagggccaca ttgactgtag acgaatcctc cagcacagtc 300

tacatgcaac tcagcagcct gacatctgag gactctgcgg tctattactg tgcaaggggg 360

ggccttcttc agtactgggg ccaaggcacc actctcacag tctcctcagc caaaacgaca 420

cccccatctg tctatccact ggcccctgga tctgctgccc aaactaactc catggtgacc 480

ctgggatgcc tggtcaaggg ctatttccct gagccagtga cagtgacctg gaactctgga 540

tccctgtcca gcggtgtgca caccttccca gctgtcctgc agtctgacct ctacactctg 600

agcagctcag tgactgtccc ctccagcacc tggcccagcg agaccgtcac ctgcaacgtt 660

gcccacccgg ccagcagcac caaggtggac aagaaaattg tgcccaggga ttgtcatcat 720

caccatcacc atcactaa 738

<210>55

<211>711

<212>DNA

<213> mouse

<400>55

atgaaatacc tattgcctac ggcagccgct ggattgttat tactcgctgc ccaaccagcc 60

atggccgaca ttcagctgac ccagtctcca gcctccctat ctgcatctgt gggagaaact 120

gtcaccatca catgtcgagc aagtgaaaac atttacagtt atttagcatg gtatcagcag 180

aaacagggaa aatctcctca gctcctggtc tataatacaa aaaccttagg agaaggtgtg 240

ccatcaaggt tcagtggcag tggatcgggc acacaatttt ctctgaagat caacagcctg 300

cagcctgaag attttgggag ttattactgt caacatcatt atggtactcc attcacgttc 360

ggctcgggga caaagttgga aataaaacgg gctgatgctg caccaactgt atccatcttc 420

ccaccatcca gtgagcagtt aacatctgga ggtgcctcag tcgtgtgctt cttgaacaac 480

ttctacccca aagacatcaa tgtcaagtgg aagattgatg gcagtgaacg acaaaatggc 540

gtcctgaaca gttggactga tcaggacagc aaagacagca cctacagcat gagcagcacc 600

ctcacgttga ccaaggacga gtatgaacga cataacagct atacctgtga ggccactcac 660

aagacatcaa cttcacccat tgtcaagagc ttcaacagga atgagtctta a 711

<210>56

<211>11

<212>PRT

<213> mouse

<400>56

Gly Tyr Thr Phe Ser Asp Tyr Trp Val His Trp

1 5 10

<210>57

<211>17

<212>PRT

<213> mouse

<400>57

Gly Ala Ile Asp Gly Ser Asp Thr Phe Asn Asp Tyr Ser Gln Lys Phe

1 5 10 15

Lys

<210>58

<211>8

<212>PRT

<213> mouse

<400>58

Ala Arg Gly Gly Leu Leu Gln Tyr

1 5

<210>59

<211>11

<212>PRT

<213> mouse

<400>59

Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala

1 5 10

<210>60

<211>7

<212>PRT

<213> mouse

<400>60

Asn Thr Lys Thr Leu Gly Glu

1 5

<210>61

<211>9

<212>PRT

<213> mouse

<400>61

Gln His His Tyr Gly Thr Pro Phe Thr

1 5

<210>62

<211>33

<212>DNA

<213> mouse

<400>62

ggctacacat tctctgacta ctgggtacac tgg 33

<210>63

<211>51

<212>DNA

<213> mouse

<400>63

ggagcgattg atggttctga tacttttaat gactacagtc agaagtttaa g 51

<210>64

<211>24

<212>DNA

<213> mouse

<400>64

gcaagggggg gccttcttca gtac 24

<210>65

<211>33

<212>DNA

<213> mouse

<400>65

cgagcaagtg aaaacattta cagttattta gca 33

<210>66

<211>21

<212>DNA

<213> mouse

<400>66

aatacaaaaa ccttaggaga a 21

<210>67

<211>27

<212>DNA

<213> mouse

<400>67

caacatcatt atggtactcc attcacg 27

<210>68

<211>33

<212>DNA

<213> mouse

<400>68

ggctacacct tcaccagcta ctggatgcac tgg 33

<210>69

<211>51

<212>DNA

<213> mouse

<400>69

ggagagattg atccttctga tagttatact aactacaatc aaaagttcaa g 51

<210>70

<211>33

<212>DNA

<213> mouse

<400>70

cgagcaagtg agaatattta cagttattta gca 33

<210>71

<211>21

<212>DNA

<213> mouse

<400>71

aatgcaaaaa ccttagcaga a 21

<210>72

<211>23

<212>DNA

<213> mouse

<400>72

caacatcatt atggtactcc tcc 23

<210>73

<211>1338

<212>DNA

<213> Artificial sequence

<220>

<223> humanized heavy chain

<400>73

caagtccaac tggttcaatc tggtgctgag gttaagaagc ctggtgcctc tgtgaaggtg 60

tcatgtaaag catctgggta cagcttcatc gagtacacca ttaattgggt ccgccaagct 120

cctggccagg gactggagtg gatcggcaat atcgatccct actacgggac cacatactac 180

aatcaaatgt tcactggcag agccaccctg accgtcgaca caagcatatc tacagcctat 240

atggagctca gccgcctgcg gtctgacgac accgctgtgt attactgcgc tcggggaagt 300

gcttggttcc catattgggg tcagggaacc ctcgttacag tctcctcagc ttcaaccaaa 360

ggccccagtg tcttccctct ggccccttcc agtaagtcta ccagcggcgg cactgccgcc 420

ctgggctgtc tcgtcaaaga ctacttccct gaacccgtga cagtgtcttg gaacagcggc 480

gcactgacaa gcggggtgca cacatttccc gccgtcctgc aatcctccgg actgtacagc 540

ctctcaagtg tggtgactgt cccatcctcc tccctcggga cccagacata tatatgcaat 600

gtgaaccata agcccagcaa caccaaggtc gataagaagg tggaacctaa aagttgcgat 660

aagactcata catgtcctcc atgccctgcc cctgaactgc tgggaggacc ttctgtcttc 720

ctgttccctc ccaagcccaa agataccctg atgatatccc gcacaccaga agtgacatgt 780

gttgttgtcg atgtctctca cgaggaccct gaagtgaagt ttaattggta tgtggacggg 840

gtggaagtgc acaacgccaa gaccaaacct cgcgaagagc agtacaactc cacataccgc 900

gtggtgagtg tgctcaccgt tctccatcag gactggctga atggcaagga gtataagtgt 960

aaggtgagca acaaagctct gccagcaccc atagagaaaa ctattagcaa agctaagggc 1020

cagcctcgcg agccacaggt gtataccctc cctcctagtc gcgaggaaat gactaagaac 1080

caggtttccc tgacatgcct cgtcaaggga ttctatccta gcgatattgc cgtcgaatgg 1140

gagtccaatg gccagcccga gaacaactac aagaccacac ctcctgtcct cgactctgac 1200

ggatccttct ttctctatag caagctgacc gttgacaaaa gcaggtggca acagggtaac 1260

gtgttttcat gctctgtgat gcacgaagcc ctgcacaatc actacacaca gaagtccctg 1320

agcctgtccc ctggcaaa 1338

<210>74

<211>446

<212>PRT

<213> Artificial sequence

<220>

<223> humanized heavy chain

<400>74

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 510 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ile Glu Tyr

20 25 30

Thr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asp Pro Tyr Tyr Gly Thr Thr Tyr Tyr Asn Gln Met Phe

50 55 60

Thr Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser Ala Trp Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210>75

<211>639

<212>DNA

<213> Artificial sequence

<220>

<223> humanized light chain

<400>75

gacattcaga tgacccaatc accaagcagt ctgtcagcca gtgttgggga tcgcgtgacc 60

ataacatgct ctgcatcctc tagtgtgact tacatgtact ggtaccaaca gaagcccggg 120

aaagccccaa agctcctgat ctatgacact agcaacctgg ctagtggagt ccccagccgg 180

ttttccggtt caggctcagg gactgactat actctcacta tttcatctct gcagcctgag 240

gactttgcca cttattattg tcagcaatgg agcaattacc cactgacctt tgggcagggc 300

accaaggtgg aaatcaagag aactgttgct gctccctccg tgttcatctt cccaccaagc 360

gatgagcagc tgaaatccgg gacagcctct gtggtgtgtc tcctgaacaa cttctatcct 420

cgggaggcaa aggtccagtg gaaagtcgat aatgccctcc agagtggcaa ctcacaagaa 480

agcgtgactg aacaggactc caaagatagt acatatagcc tcagcagtac actgaccctg 540

agcaaagccg attatgagaa acataaggtg tacgcttgcg aggtcaccca ccagggcctg 600

tccagtccag tgactaagag ctttaataga ggtgagtgt 639

<210>76

<211>213

<212>PRT

<213> Artificial sequence

<220>

<223> humanized light chain

<400>76

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Thr Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Asn Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

Claims (25)

1. A pharmaceutical combination comprising:

(A) (ii) (i) an anti-BST 1 antibody, or antigen-binding portion thereof, that competes for binding to BST1 with an antibody having a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4;

or

(ii) An anti-BST 1 antibody or antigen-binding portion thereof, the antibody or portion comprising:

a) a heavy chain variable region comprising:

i) a first vhCDR comprising SEQ ID NO 10;

ii) a second vhCDR comprising a sequence selected from SEQ ID NO 12 and SEQ ID NO 51; and

iii) a third vhCDR comprising SEQ ID NO 14; and

b) a light chain variable region comprising:

i) a first vlCDR comprising SEQ ID NO 16;

ii) a second vlCDR comprising SEQ ID NO 18; and

iii) a third vlCDR comprising SEQ ID NO 20;

optionally, wherein any one or more of the above SEQ ID NOs each independently comprises one, two, three, four, or five amino acid substitutions, additions, or deletions;

and

(B) a cytidine analog, or a pharmaceutically acceptable salt thereof,

wherein the pharmaceutical combination is in the form of a combined preparation for simultaneous, separate or sequential use.

2. The pharmaceutical combination according to claim 1, wherein the use is for the treatment of cancer.

3. The pharmaceutical combination according to claim 1 or 2, wherein the cytidine analog is 5-aza-cytidine or 5-aza-2' -deoxycytidine.

4. The pharmaceutical combination of any one of claims 1 to 3, wherein any one or more of SEQ ID NOs 10, 12, 51, 14, 16, 18 or 20 each independently comprises one, two, three, four or five conservative amino acid substitutions.

5. The pharmaceutical combination of claim 4, wherein any one or more of SEQ ID NOs 10, 12, 51, 14, 16, 18 or 20 each independently comprises one or two conservative amino acid substitutions.

6. The pharmaceutical combination of any one of claims 1 to 5, wherein the anti-BST 1 antibody, or antigen-binding portion thereof, comprises:

(a) a heavy chain variable region having at least 80%, 85%, 90%, 95%, 99% or 100% amino acid sequence identity to SEQ ID NO 2 or SEQ ID NO 46, and

(b) a light chain variable region having at least 80%, 85%, 90%, 95%, 99% or 100% amino acid sequence identity to SEQ ID NO. 4 or SEQ ID NO. 49.

7. The pharmaceutical combination of any one of claims 1 to 6, wherein the anti-BST 1 antibody comprises:

(a) a heavy chain having at least 80%, 85%, 90%, 95%, 99% or 100% amino acid sequence identity to SEQ ID NO 74, and

(b) a light chain having at least 80%, 85%, 90%, 95%, 99%, or 100% amino acid sequence identity to SEQ ID NO. 76.

8. The pharmaceutical combination of any one of claims 1 to 7, wherein the anti-BST 1 antibody is a human IgG1 monoclonal antibody.

9. The pharmaceutical combination of any one of claims 1 to 8, wherein the anti-BST 1 antibody induces antibody dependent cell mediated cytotoxicity (ADCC), Complement Dependent Cytotoxicity (CDC) and/or T cell cytotoxicity, preferably ADCC.

10. The pharmaceutical combination of claim 9, wherein the anti-BST 1 antibody is an engineered antibody with enhanced binding to Fc receptors and/or enhanced ADCC potency, preferably the antibody is afucosylated or defucosylated.

11. The pharmaceutical combination of any one of claims 1 to 10, wherein the antibody is a bispecific or multispecific antibody that specifically binds a first antigen comprising BST1 and a second antigen selected from the group consisting of a CD3 antigen and a CD5 antigen.

12. The pharmaceutical combination of any one of claims 1 to 11, wherein (a) and/or (B) further comprises one or more pharmaceutically acceptable diluents, excipients or carriers.

13. The pharmaceutical combination according to any one of claims 1 to 12, wherein the pharmaceutical combination is in the form of a combined preparation for simultaneous, separate or sequential use in the treatment of Acute Myeloid Leukemia (AML), B-cell chronic lymphocytic leukemia, breast cancer, colorectal cancer, kidney cancer, head and neck cancer, lung cancer, ovarian cancer and pancreatic cancer, preferably Acute Myeloid Leukemia (AML).

14. The pharmaceutical combination of any one of claims 1 to 13, further comprising instructions for treating cancer in a patient in need of such treatment by administering (a) and (B) to said patient.

15. A kit, comprising:

(i) an anti-BST 1 antibody or antigen-binding portion thereof as defined in any one of claims 1 or 4 to 7; and

(ii) cytidine analogs, preferably 5-aza-cytidine or 5-aza-2' -deoxycytidine, or a pharmaceutically acceptable salt thereof.

16. A method of treating cancer in a patient, comprising administering to a patient in need thereof simultaneously, sequentially or separately therapeutically effective amounts of components (a) and (B) of a pharmaceutical combination as defined in any one of claims 1 to 13.

17. The method of claim 16, wherein the anti-BST 1 antibody or antigen-binding portion is afucosylated or defucosylated.

18. The method of claim 16 or 17, wherein the cancer is Acute Myeloid Leukemia (AML), B-cell chronic lymphocytic leukemia, breast cancer, colorectal cancer, kidney cancer, head and neck cancer, lung cancer, ovarian cancer and pancreatic cancer, preferably Acute Myeloid Leukemia (AML).

19. A pharmaceutical combination according to any one of claims 1 to 13 for use in the treatment of cancer, wherein components (a) and (B) are administered to a patient simultaneously, separately or sequentially for use in the treatment of cancer.

20. The pharmaceutical combination for use according to claim 19, wherein the anti-BST 1 antibody or antigen-binding portion is afucosylated or defucosylated.

21. The pharmaceutical combination for use according to claim 19 or 20, wherein the cancer is Acute Myeloid Leukemia (AML), B-cell chronic lymphocytic leukemia, breast cancer, colorectal cancer, kidney cancer, head and neck cancer, lung cancer, ovarian cancer and pancreatic cancer, preferably Acute Myeloid Leukemia (AML).

22. Use of components (a) and (B) of a pharmaceutical combination as defined in any one of claims 1 to 13 for the preparation of a pharmaceutical combination for simultaneous, separate or sequential use in the treatment of cancer.

23. The use of claim 22, wherein the anti-BST 1 antibody or antigen-binding portion is afucosylated or defucosylated.

24. The use according to claim 22 or 23, wherein the cancer is Acute Myeloid Leukemia (AML), B-cell chronic lymphocytic leukemia, breast cancer, colorectal cancer, kidney cancer, head and neck cancer, lung cancer, ovarian cancer and pancreatic cancer, preferably Acute Myeloid Leukemia (AML).

25. A pharmaceutical combination according to any one of claims 1 to 13 for use in therapy or as a medicament.

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